Cell-specific adenovirus vectors comprising an internal ribosome entry site

ABSTRACT

Disclosed herein are replication-competent adenovirus vectors comprising co-transcribed first and second genes under transcriptional control of a heterologous, target cell-specific transcriptional regulatory element (TRE), wherein the second gene is under translational control of an internal ribosome entry site. Methods for the preparation and use of such vectors are also provided. The vectors provide target cell-specific virus replication in applications such as cancer therapy and gene therapy.

TECHNICAL FIELD

[0001] This invention relates to new replication competent adenovirusvectors comprising an internal ribosome entry site which replicatepreferentially in target cells. The present invention also relates tocell transduction using adenovirus vectors comprising an internalribosome entry site.

BACKGROUND

[0002] Diseases involving altered cell proliferation, particularlyhyperproliferation, constitute an important health problem. For example,despite numerous advances in medical research, cancer remains the secondleading cause of death in the United States. In the industrializednations, roughly one in five persons will die of cancer. Traditionalmodes of clinical care, such as surgical resection, radiotherapy andchemotherapy, have a significant failure rate, especially for solidtumors. Neoplasia resulting in benign tumors can usually be completelycured by surgical removal of the tumor mass. If a tumor becomesmalignant, as manifested by invasion of surrounding tissue, it becomesmuch more difficult to eradicate. Once a malignant tumor metastasizes,it is much less likely to be eradicated.

[0003] Excluding basal cell carcinoma, there are over one million newcases of cancer per year in the United States alone, and cancer accountsfor over one half million deaths per year in this country. In the worldas a whole, the five most common cancers are those of lung, stomach,breast, colon/rectum, and uterine cervix, and the total number of newcases per year is over 6 million.

[0004] In the United States, transitional cell carcinoma (TCC) accountsfor 90 to 95 percent of all tumors of the bladder. Squamous cellcarcinoma (SCC) represents 5 to 10 percent, and adenocarcinomaapproximately 1 to 2 percent. Squamous cell and adenomatous elements areoften found in association with transitional cell tumors, especiallywith high grade tumors. Bladder cancer is generally divided intosuperficial and invasive disease. A critical factor is the distinctionbetween those tumors that are confined to the mucosa and those that havepenetrated the basement membrane and extended into the lamina propria.The term “superficial bladder tumor” is generally used to represent atumor that has not invaded the muscularis. Invasive tumors are describedas those that have invaded the muscularis propria, the perivesicalfibroadipose tissue, or adjacent structures. Carcinoma in situ (CIS) isa high grade and aggressive manifestation of TCC of the bladder that hasa highly variable course.

[0005] A number of urothelial cell-specific proteins have beendescribed, among which are the uroplakins. Uroplakins (UP), includingUPIa and UPIb (27 and 28 kDa, respectively), UPII (15 kDa), and UPIII(47 kDa), are members of a group of integral membrane proteins that aremajor proteins of urothelial plaques. These plaques cover a largeportion of the apical surface of mammalian urothelium and may play arole as a permeability barrier and/or as a physical stabilizer of theurothelial apical surface. Wu et al. (1994) J. Biol. Chem.269:13716-13724. UPs are bladder-specific proteins, and are expressed ona significant proportion of urothelial-derived tumors, including about88% of transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol.147:1383-1397; and Wu et al. (1998) Cancer Res. 58:1291-1297. Thecontrol of the expression of the human UPII has been studied, and a3.6-kb region upstream of the mouse UPII gene has been identified whichcan confer urothelial-specific transcription on heterologous genes (Linet al. (1995) Proc. Natl. Acad. Sci. USA 92:679-683). See also, U.S.Pat. Nos. 5,824,543 and 6,001,646.

[0006] Melanoma, a malignant neoplasm derived from melanocytes of theskin and other sites, has been increasing in incidence worldwide. TheAmerican Joint Committee on Cancer recognizes five different forms ofextraocular melanoma occurring in humans: lentigo maligna melanoma;radial spreading; nodular; acral lentiginous; and unclassified. Knownmelanoma-associated antigens can be classified into three main groups:tumor-associated testis-specific antigens MAGE, BAGE, GAGE, and PRAME;melanocyte differentiation antigens tyrosinase, Melan-A/MART-1 (forMelanoma Antigen Recognized by T cells), gp100, tyrosinase relatedprotein-1 (TRP-1), tyrosinase related protein-2 (TRP-2); and mutated oraberrantly expressed antigens MUM-1, cyclin-dependent kinase 4 (CDK4),beta-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V.See, for example, Kirkin et al. (1998) Exp. Clin. Immunogenet. 15:19-32.Tyrosinase, TRP-1, and TRP-2 are enzymes involved in melaninbiosynthesis and are specifically expressed in melanocytes. Antigenicepitopes of MART-1 have been studied extensively, with the aim ofdeveloping a melanoma vaccine. An immunodominant epitope, MART-1(27-35)has been reported to be recognized by a majority of CD8+ cytotoxic Tcell clones generated to MART-1. These MART-[(27-35)-specific CTLsspecifically lyse autologous tumor cell lines expressing the epitope.Faure and Kourilsky (1998) Crit. Rev. Immunol. 18:77-86. However, othershave reported that presence of such CTLs is not accompanied by asignificant clinical response. Rivoltini et al. (1998) Crit. Rev.Immunol. 18:55-63.

[0007] A major, indeed the overwhelming, obstacle to cancer therapy isthe problem of selectivity; that is, the ability to inhibit themultiplication of tumor cells without affecting the functions of normalcells. For example, in traditional chemotherapy of prostate cancer, thetherapeutic ratio, (i.e., the ratio of tumor cell killing to normal cellkilling) is only 1.5:1. Thus, more effective treatment methods andpharmaceutical compositions for therapy and prophylaxis of neoplasia areneeded.

[0008] Accordingly, the development of more specific, targeted forms ofcancer therapy, especially for cancers that are difficult to treatsuccessfully, is of particular interest. In contrast to conventionalcancer therapies, which result in relatively non-specific and oftenserious toxicity, more specific treatment modalities, which inhibit orkill malignant cells selectively while leaving healthy cells intact, arerequired.

[0009] Gene therapy, whereby a gene of interest is introduced into amalignant cell, has been attempted as an approach to treatment of manycancers. See, for example, Boulikas (1997) Anticancer Res. 17:1471-1505,for a description of gene therapy for prostate cancer. A gene ofinterest can encode a protein which is converted into a toxic substanceupon treatment with another compound, or it can encode an enzyme thatconverts a prodrug to a drug. For example, introduction of the herpessimplex virus gene encoding thymidine kinase (HSV-tk) renders cellsconditionally sensitive to ganciclovir. Zjilstra et al. (1989) Nature342: 435; Mansour et al. (1988) Nature 336: 348; Johnson et al. (1989)Science 245: 1234; Adair et al. (1989) Proc. Natl. Acad. Sci. USA 86:4574; Capecchi (1989) Science 244: 1288. Alternatively, a gene ofinterest can encode a compound that is directly toxic, such as, forexample, diphtheria toxin. To render these treatments specific to cancercells, the gene of interest is placed under control of a transcriptionalregulatory element (TRE) that is specifically (i.e., preferentially)active in the cancer cells. Cell- or tissue-specific expression can beachieved by using a TRE with cell-specific enhancers and/or promoters.See generally Huber et al. (1995) Adv. Drug Delivery Reviews 17:279-292.

[0010] A number of viral vectors and non-viral delivery systems (e.g.,liposomes), have been developed for gene transfer. Of the virusesproposed for gene transfer, adenoviruses are among the most easilyproduced and purified. Adenovirus also has the advantage of a highefficiency of transduction (i.e., introduction of the gene of interestinto the target cell) and does not require cell proliferation forefficient transduction. In addition, adenovirus can infect a widevariety of cells in vitro and in vivo. For general background referencesregarding adenovirus and development of adenoviral vector systems, seeGraham et al. (1973) Virology 52:456-467; Takiff et al. (1981) Lancet11:832-834; Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020;Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806.

[0011] Adenoviruses generally undergo a lytic replication cyclefollowing infection of a host cell. In addition to lysing the infectedcell, the replicative process of adenovirus blocks the transport andtranslation host cell mRNA, thus inhibiting cellular protein synthesis.For a review of adenoviruses and adenovirus replication, see Shenk, T.and Horwitz, M. S., Virology, third edition, Fields, B. N. et al., eds.,Raven Press Limited, New York (1996), Chapters 67 and 68, respectively.

[0012] When used for gene transfer, adenovirus vectors are oftendesigned to be replication-defective and are thus deliberatelyengineered to fail to replicate in the target cell. In these vectors,the early adenovirus gene products E1A and/or E1B are often deleted, andthe gene to be transduced is commonly inserted into the E1A and/or E1Bregion of the deleted virus genome. Bett et al. (1994) supra. Suchvectors are propagated in packaging cell lines such as the 293 line,which provides E1A and E1B functions in trans. Graham et al. (1987) J.Gen. Virol 36:59-72; Graham (1977) J. Gen. Virol. 68:937-940. The use ofreplication-defective adenovirus vectors as vehicles for efficienttransduction of genes has been described by, inter alia,Stratford-Perricaudet (1990) Human Gene Therapy 1:241-256; Rosenfeld(1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol.309:61-66; Jaffe et al. (1992) Nature Gen. 1:372-378; Quantin et al.(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992)Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest.90:626-630; Le Gal Le Salle et al. (1993) Science 259:988-990;Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al.(1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem.269:23872-23875; and Bett et al. (1994) supra.

[0013] In the treatment of cancer by replication-defective adenoviruses,the host immune response limits the duration of repeat doses at twolevels. First, the capsid proteins of the adenovirus delivery vehicleitself are immunogenic. Second, viral late genes are frequentlyexpressed in transduced cells, eliciting cellular immunity. Thus, theability to repeatedly administer cytokines, tumor suppressor genes,ribozymes, suicide genes, or genes which convert a prodrug to an activedrug has been limited by the immunogenicity of both the gene transfervehicle and the viral gene products of the transfer vehicle, coupledwith the transient nature of gene expression. Despite these limitations,development of adenoviral vectors for gene therapy has focused almostexclusively on the use of the virus as a vehicle for introducing a geneof interest, not as an effector in itself. In fact, replication ofadenovirus vectors has been viewed as an undesirable result, largely dueto the host immune response.

[0014] More recently, however, the use of adenovirus vectors aseffectors has been described. International Patent Application Nos.PCT/US98/04080, PCT/US98/04084, PCT/US98/04133, PCT/US98/04132,PCT/US98/16312, PCT/US95/00845, PCT/US96/10838, PCT/EP98/07380 and U.S.Pat. No. 5,998,205. Adenovirus E1A and E1B genes are disclosed in Rao etal. (1992, Proc. Natl. Acad. Sci. USA vol. 89: 7742-7746).

[0015] Replication-competent adenovirus vectors, which take advantage ofthe cytotoxic effects associated with adenovirus replication, haverecently been described as agents for effecting selective cell growthinhibition. In such systems, a cell-specific transcriptional regulatoryelement (TRE) is used to control the expression of a gene essential forviral replication, thus limiting viral replication to cells in which theTRE is functional. See, for example International Patent Application No.PCT/EP99/07380, Henderson et al., U.S. Pat. No. 5,698,443; Hallenbeck etal., PCT/US95/15455 and U.S. Pat. No. 5,998,205; Rodriguez et al. (1997)Cancer Res. 57:2559-2563.

[0016] PCT publication PCT/US98/04080 discloses replication-competent,target cell-specific adenovirus vectors comprising heterologous TREs,such as those regulating expression of prostate-specific antigen (PSA),probasin (PB), α-fetoprotein (AFP), kallikrien (hKLK2), mucin (MUC1) andcarcinoembryonic antigen (CEA). PCT/US98/04084 disclosesreplication-competent adenovirus vectors comprising an α-fetoprotein(AFP) TRE that replicate specifically in cells expressing AFP, such ashepatoma cells.

[0017] Internal ribosome entry sites (IRES) are sequences which initiatetranslation from an internal initiation codon (usually AUG) within abi-or multi-cistronic RNA transcript continuing multiple protein codingregions. IRES have been characterized in encephalomyocarditis virus andrelated picornaviruses. See, for example, Jackson et al. (1995) RNA 1:985-1000 and Herman (1989) Trends in Biochemical Sciences 14(6):219-222. IRES sequences are also detected in mRNAs from other virusessuch as cardiovirus, rhinovirus, aphthovirus, hepatitis C virus (HCV),Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus(MoMLV). The presence of IRES in cellular RNAs has also been described.Examples of cellular mRNAs containing IRES include those encodingimmunoglobulin heavy-chain binding protein (BiP), vascular endothelialgrowth factor (VEGF), fibroblast growth factor 2, insulin-like growthfactor, translational initiation factor eIF4G, and the yeasttranscription factors TFIID and HAP4. See, for example, Macejak et al.(1991) Nature 353:90-94; Oh et al. (1992) Genes Dev. 6:1643-1653; Vagneret al. (1995) Mol. Cell. Biol. 15:35-44; He et al. (1996) Proc. Natl.Acad. Sci USA 93:7274-7278; He et al. (1996) Gene 175:121-125; Tomaninet al. (1997) Gene 193:129-140; Gambotto et al. (1999) Cancer GeneTherapy 6:45-53; Qiao et al. (1999) Cancer Gene Therapy 6:373-379.Expression vectors containing IRES elements have been described. See,for example, International Patent Application No. PCT/US98/03699 andInternational Patent Application No. PCT/EP98/07380.

[0018] Thus, there is a continuing need for improvedreplication-competent adenovirus vectors in which cell-specificreplication can be further enhanced, while minimizing the extent ofreplication in non-target (i.e., non-cancerous cells).

[0019] The disclosure of all patents and publications cited herein areincorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0020] The present invention provides improved replication competentadenovirus vectors comprising co-transcribed first and second genesunder transcriptional control of a heterologous, target cell-specifictranscriptional regulatory element (TRE), wherein the second gene isunder translational control of an internal ribosome entry site (IRES).In one embodiment, the first and second genes are co-transcribed as asingle mRNA and the second gene has a mutation in or deletion of itsendogenous promoter. The present invention further provides host cellsand methods using the adenovirus vectors.

[0021] In one aspect, the first and/or second genes are adenovirus genesand in another aspect, the first and/or second adenovirus genes areessential for viral replication. An essential gene can be an early viralgene, including for example, E1A; E1B; E2; and/or E4, or a late viralgene. In another aspect an early gene is E3.

[0022] In one embodiment, the first gene is an adenovirus gene and thesecond gene is a therapeutic gene. In another embodiment, both genes areadenovirus genes. In an additional embodiment, the first adenovirus geneis E1A, and the second adenovirus gene is E1B. Optionally, theendogenous promoter for one of the co-transcribed adenovirus geneessential for viral replication, such as for example, E1A, is deletedand/or mutated such that the gene is under sole transcriptional controlof a target cell-specific TRE.

[0023] In another aspect, the present invention provides adenovirusvectors comprising an adenovirus gene essential for viral replicationunder control of a target cell-specific TRE, wherein said adenovirusgene has a mutation of or deletion in its endogenous promoter. In oneembodiment, the adenovirus gene is essential for viral replication. Inanother embodiment, the adenovirus gene is E1A wherein the E1A promoteris deleted and wherein the E1A gene is under transcriptional control ofa heterologous cell-specific TRE. In another embodiment, the adenovirusgene is E1B wherein the E1B promoter is deleted and wherein the E1B geneis under transcriptional control of a heterologous cell-specific TRE.

[0024] In another aspect, the present invention provides adenovirusvectors comprising E1B under control of a target cell-specific TRE,wherein said E1B has a deletion in or mutation of the 19-kDa region ofE1B, that encodes a product shown to inhibit apoptosis.

[0025] In other embodiments, an enhancer element for the first and/orsecond adenovirus genes is inactivated. The present invention providesan adenovirus vector comprising E1A wherein an E1A enhancer isinactivated. In yet other embodiments, the present invention provides anadenovirus vector comprising E1A wherein the E1A promoter is inactivatedand E1A enhancer I is inactivated. In further embodiments, the presentinvention provides an adenovirus vector comprising a TRE which has itsendogenous silencer element inactivated.

[0026] Any TRE which directs cell-specific expression can be used in thedisclosed vectors. In one embodiment, TREs include, for example, TREsspecific for prostate cancer cells, breast cancer cells, hepatoma cells,melanoma cells, bladder cells and/or colon cancer cells. In anotherembodiment, the TREs include, probasin (PB) TRE; prostate-specificantigen (PSA) TRE; mucin (MUC1) TRE; α-fetoprotein (AFP) TRE; hKLK2 TRE;tyrosinase TRE; human uroplakin II TRE (hUPII) and carcinoembryonicantigen (CEA) TRE. In other embodiments, the target cell-specific TRE isa cell status-specific TRE. In yet other embodiments, the targetcell-specific TRE is a tissue specific TRE.

[0027] In additional embodiments, the adenovirus vector comprises atleast one additional co-transcribed gene under the control of thecell-specific TRE. In another embodiment, an additional co-transcribedgene is under the translational control of an IRES.

[0028] In another aspect of the present invention, adenovirus vectorsfurther comprise a transgene such as, for example, a cytotoxic gene. Inone embodiment, the transgene is under the transcriptional control ofthe same TRE as the first gene and second genes and optionally under thetranslational control of an internal ribosome entry site. In anotherembodiment, the transgene is under the transcriptional control of adifferent TRE that is functional in the same cell as the TRE regulatingtranscription of the first and second genes and optionally under thetranslational control of an IRES.

[0029] The present invention also provides compositions comprising thereplication-competent adenovirus vectors described herein. In oneembodiment, the compositions further comprise a pharmaceuticallyacceptable excipient. The present invention also provides kitscomprising the replication-competent adnenovirus vectors describedherein.

[0030] Host cells comprising the disclosed adenovirus vectors are alsoprovided. Host cells include those used for propagation of a vector andthose into which a vector is introduced for therapeutic purposes.

[0031] In another aspect, methods are provided for propagatingreplication-competent adenovirus vectors of the present inventionspecific for mammalian cells which permit the function of a targetcell-specific TRE, said method comprising combining an adenovirusvector(s) described herein with mammalian cells that permit the functionof a target cell-specific TRE, such that the adenovirus vector(s) entersthe cell, whereby said adenovirus is propagated.

[0032] In another aspect, methods are provided for conferring selectivecytotoxicity in target cells, comprising contacting the cells with anadenovirus vector(s) described herein, whereby the vector enters thecell.

[0033] The invention further provides methods of suppressing tumor cellgrowth, more particularly a target tumor cell, comprising contacting atumor cell with an adenovirus vector(s) of the invention such that theadenovirus vector enters the tumor cell and exhibits selectivecytotoxicity for the tumor cell.

[0034] In another aspect, methods are provided for detecting a cellwhich allows the function of a target cell-specific TRE, which comprisecontacting a cell in a biological sample with an adenovirus vector(s) ofthe invention, and detecting replication of the adenovirus vector(s), ifany.

[0035] In another aspect, methods are provided for modifying thegenotype of a target cell, comprising contacting the cell with anadenovirus vector as described herein, wherein the adenovirus vectorenters the cell.

[0036] The present invention provides an adenovirus vector comprising anadenovirus gene, wherein said adenovirus gene is under transcriptionalcontrol of a melanocyte-specific TRE. In another embodiment, amelanocyte-specific TRE is human. In another embodiment, amelanocyte-specific TRE comprises a melanocyte-specific promoter and aheterologous enhancer. In other embodiments, a melanocyte-specific TREcomprises a melanocyte-specific promoter. In other embodiments, amelanocyte-specific TRE comprises a melanocyte-specific enhancer and aheterologous promoter. In other embodiments, a melanocyte-specific TREcomprises a melanocyte-specific promoter and a melanocyte-specificenhancer.

[0037] In some embodiments, the adenovirus gene under transcriptionalcontrol of a melanocyte-specific TRE is an adenovirus gene essential forreplication. In some embodiments, the adenoviral gene essential forreplication is an early gene. In another embodiment, the early gene isE1A. In another embodiment, the early gene is E1B. In yet anotherembodiment, both E1A and E1B are under transcriptional control of amelanocyte-specific TRE. In further embodiments, the adenovirus geneessential for replication is E1B, and E1B has a deletion in the 19-kDaregion.

[0038] In some embodiments, the melanocyte-specific TRE is derived fromthe 5′ flanking region of a tyrosinase gene. In other embodiments, themelanocyte-specific TRE is derived from the 5′ flanking region of atyrosinase related protein-1 gene. In other embodiments, themelanocyte-specific TRE is derived from the 5′-flanking region of atyrosinase related protein-2 gene. In other embodiments, themelanocyte-specific TRE is derived from the 5′ flanking region of aMART-1 gene. In other embodiments, the melanocyte-specific TRE isderived from the 5′-flanking region of a gene which is aberrantlyexpressed in melanomas.

[0039] In other embodiments, the invention provides an adenovirus vectorcomprising (a) an adenovirus gene under transcriptional control of amelanocyte-specific TRE; and (b) an E3 region. In some of theseembodiments the E3 region is under transcriptional control of amelanocyte-specific TRE.

[0040] In another aspect, the invention provides a host cell comprisingthe melanocyte specific adenovirus vector(s) described herein.

[0041] In another aspect, the invention provides pharmaceuticalcompositions comprising a melanocyte specific adenovirus vector(s)described herein.

[0042] In another aspect, the invention provides kits which contain amelanocyte adenoviral vector(s) described herein.

[0043] In another aspect, methods are provided for conferring selectivecytoxicity in target cells (i.e., cells which permit or induce amelanocyte-specific TRE to function), comprising contacting the cellswith an adenovirus vector(s) described herein, whereby the vector entersthe cell.

[0044] In another aspect, methods are provided for propagating anadenovirus specific for melanocytes, said method comprising combining anmelanocyte specific adenovirus vector(s) described herein withmelanocytes, whereby said adenovirus is propagated.

[0045] The invention further provides methods of suppressing melanomacell growth, comprising contacting a melanoma cell with a melanocytespecific adenoviral vector of the invention such that the adenoviralvector enters the melanoma cell and exhibits selective cytotoxicity forthe melanoma cell.

[0046] In another aspect, methods are provided for detectingmelanocytes, including melanoma cells, in a biological sample,comprising contacting cells of a biological sample with a melanocyteadenovirus vector(s) described herein, and detecting replication of theadenovirus vector, if any.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1 is a schematic of plasmid construct CP627 as described inExample 1.

[0048] FIGS. 2A-2B is a series of schematic depictions of variousadenoviruses described herein.

[0049]FIG. 3 depicts the replication efficiency of different viruses asdescribed in Example 4.

[0050]FIGS. 4A and 4B show viral yield for different liver-specificvectors in different cell types.

[0051]FIG. 5 is a schematic representation of adenovirus vectorscomprising AFP-TRE with and without IRES.

[0052]FIG. 6 depicts an E3 region.

[0053]FIG. 7 is a schematic representation of adenovirus vectorsdescribed herein.

[0054]FIG. 8 depicts in vivo antitumor activity of CV890 containing anIRES. This figure depicts the results of a HepG2 Xenograph treated withCV790 or CV890.

[0055]FIG. 9 depicts an ADP nucleotide and amino acid sequence.

[0056]FIG. 10 depicts an IC₅₀ isobologram of doxorubicin and CV 890 onHep3B cells at day 5.

[0057]FIG. 11 depicts in vivo efficacy of CV890 with doxorubicin. Hep3Bnude mouse xenografts were grouped (n=6) and treated with CV890 alone(1×10¹¹ particles/dose, iv), doxorubicin alone (10 mg/kg, ip), CV890 anddoxorubicin combination (1×10¹¹ particles of CV890 through tail vein and10 mg/kg doxorubicin ip), or vehicle control. Tumor size was measuredweekly and the tumor volume were normalized as 100% at the day oftreatment. Error bars represent the standard error of the mean.

[0058]FIG. 12 shows the virus yield of CV802, CV882 and CV884 in celllines.

MODES FOR CARRYING OUT THE INVENTION

[0059] We have discovered and constructed improved adenovirus vectorscomprising co-transcribed first and second genes under transcriptionalcontrol of a heterologous, target cell-specific transcriptionalregulatory element (TRE), wherein the second gene is under translationalcontrol of an internal ribosome entry site (IRES). In one embodiment,the first and second genes are co-transcribed as a single mRNA and thesecond gene has a mutation in or deletion of its endogenous promoter. Inanother embodiment, at least one of the genes is an adenovirus gene andin yet another embodiment, both genes are adenovirus genes, includingadenovirus genes that are essential for viral replication. Theadenovirus vector may comprise a gene that contributes to cytotoxicity(whether direct and/or indirect), and/or causes cell death. An exampleof an adenovirus gene that contributes to cytotoxicity includes, but isnot limited to, the adenovirus death protein gene.

[0060] In some aspects of the present invention, an adenovirus vectorcomprising co-transcribed first and second genes under transcriptionalcontrol of a target cell-specific TRE, wherein the second gene is undertranslational control of an IRES, exhibits greater specificity for thetarget cell than an adenovirus vector comprising a target cell-specificTRE operably linked to a gene and lacking an IRES. In some embodiments,specificity is conferred by preferential transcription and/ortranslation of the first and second genes due to the presence of atarget cell specific TRE. In other embodiments, specificity is conferredby preferential replication of the adenovirus vectors in target cellsdue to the target cell-specific TRE driving transcription of a geneessential for replication.

[0061] Also disclosed herein are IRES containing adenovirus vectorscomprising an adenovirus gene essential for viral replication whereinsaid essential gene has a mutation in or deletion of its endogenouspromoter. In an embodiment disclosed herein, the adenovirus vectorscomprise the adenovirus early gene E1A which has a deletion of itsendogenous promoter. In another embodiment disclosed herein, theadenovirus vectors comprise the adenovirus early gene E1B which has adeletion of its endogenous promoter. In other embodiments disclosedherein, the 19-kDa region of E1B is deleted.

[0062] In another aspect, the adenovirus vectors disclosed hereincomprise an adenovirus gene essential for viral replication wherein saidessential gene has a mutation in or deletion of its endogenous enhancer.In one embodiment, the adenovirus vector comprises the adenovirus earlygene E1A which has a mutation of or deletion in its endogenous promoter.In one embodiment, the adenovirus vector comprises the adenovirus earlygene E1A which has a mutation of or deletion in E1A enhancer 1. In afurther embodiment, the adenovirus vector comprises the adenovirus earlygene E1A which has a mutation of or deletion in its endogenous promoterand a mutation of or deletion in the E1A enhancer. In an additionalembodiment, the adenovirus vector comprises the adenovirus early geneE1A which has a mutation of or deletion in its endogenous promoter andthe adenovirus early gene E1B which has a mutation of or deletion in itsendogenous promoter. In an additional embodiment, the adenovirus vectorcomprises the adenovirus early gene E1A, which has a mutation of ordeletion in its endogenous promoter and a mutation of or deletion in theE1A enhancer I, and the adenovirus early gene E1B which has a mutationof or deletion in its endogenous promoter. In other embodimentsdisclosed herein, the 19-kDa region of E1B is deleted.

[0063] The replication-competent adenovirus vectors of the presentinvention take advantage of what has been heretofore considered anundesirable aspect of adenovirus vectors, namely their replication andpossible concomitant immunogenicity. Runaway infection is prevented dueto the cell-specific requirements for viral replication. Without wishingto be bound by any particular theory, it is noted that production ofadenovirus proteins can serve to activate and/or stimulate the immunesystem, either generally or specifically, toward target cells producingadenoviral proteins. This type of immune stimulation can be an importantconsideration in the cancer context, where patients are often moderatelyto severely immunocompromised.

[0064] The adenovirus vectors of the present invention comprising anintergenic IRES element(s) which links the translation of two or moregenes, reflects an improvement over vector constructs which useidentical control regions to drive expression of two or more desiredgenes in that any potential for homologous recombination based on thepresence of homologous control regions in the vector is removed. Asdemonstrated herein, adenovirus vectors comprising an IRES are stableand in some embodiments provide better specificity than vectors notcontaining an IRES. Another advantage of an adenovirus vector comprisingan intergenic IRES is that the use of an IRES rather than a second TREmay provide additional space in the vector for an additional gene(s)such as a therapeutic gene.

[0065] Thus, the adenovirus vectors comprising a second gene undercontrol of an IRES retain a high level of target cell specificity andremain stable in the target cell. Accordingly, in one aspect of theinvention, the viral vectors disclosed herein comprise at least one IRESwithin a multicistronic transcript, wherein production of themulticistronic transcript is regulated by a heterologous, targetcell-specific TRE. For adenovirus vectors comprising a second gene undercontrol of an IRES, it is preferred that the endogenous promoter of agene under translational control of an IRES be deleted so that theendogenous promoter does not interfere with transcription of the secondgene. It is preferred that the second gene be in frame with the IRES ifthe IRES contains an initiation codon. If an initiation codon, such asATG, is present in the IRES, it is preferred that the initiation codonof the second gene is removed and that the IRES and the second gene arein frame. Alternatively, if the IRES does not contain an initiationcodon or if the initiation codon is removed from the IRES, theinitiation codon of the second gene is used. In one embodiment, theadenovirus vectors comprises the adenovirus essential genes, E1A and E1Bgenes, under the transcriptional control of a heterologous,cell-specific TRE, and an IRES introduced between E1A and E1B. Thus,both E1A and E1B are under common transcriptional control, andtranslation of E1B coding region is obtained by virtue of the presenceof the IRES. In one embodiment, E1A has its endogenous promoter deleted.In another embodiment, E1A has an endogenous enhancer deleted and in yetan additional embodiment, E1A has its endogenous promoter deleted andE1A enhancer I deleted. In another embodiment, E1B has its endogenouspromoter deleted. In other embodiments disclosed herein, the 19-kDaregion of E1B is deleted.

[0066] To provide cytotoxicity to target cells, one or more transgeneshaving a cytotoxic effect may be present in the vector. Additionally, oralternatively, an adenovirus gene that contributes to cytotoxicityand/or cell death, such as the adenovirus death protein (ADP) gene, canbe included in the vector, optionally under the selectivetranscriptional control of a heterologous TRE and optionally under thetranslational control of an IRES.

[0067] Examples of target cells include neoplastic cells, although anycell for which it is desirable and/or tolerable to sustain a cytotoxicactivity can be a target cell. By combining an adenovirus vector(s)comprising a target cell-specific TRE with a mixture of target andnon-target cells, in vitro or in vivo, the vector(s) preferentiallyreplicates in the target cells, causing cytotoxic and/or cytolyticeffects. Once the target cells are destroyed due to selective cytotoxicand/or cytolytic activity, replication of the vector(s) is significantlyreduced, lessening the probability of runaway infection and undesirablebystander effects. In vitro cultures can be retained to continuallymonitor the mixture (such as, for example, a biopsy or other appropriatebiological sample) for the presence of the undesirable target cell,e.g., a cancer cell in which the target cell-specific TRE is functional.The adenovirus vectors of the present invention can also be used in exvivo procedures wherein desirable biological samples comprising targetcells are removed from the animal, subjected to exposure to anadenovirus vector of the present invention comprising a targetcell-specific TRE and then replaced within the animal.

[0068] General Techniques

[0069] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal CellCulture (R. I. Freshney, ed., 1987); Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Wei & C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987 and annual updates); PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991 and annualupdates).

[0070] For techniques related to adenovirus, see, inter alia, Felgnerand Ringold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl.Acids Res. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al.(1993) J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad.Sci. USA 91:8802-8806.

[0071] Definitions

[0072] As used herein, an “internal ribosome entry site” or “IRES”refers to an element that promotes direct internal ribosome entry to theinitiation codon, such as ATG, of a cistron (a protein encoding region),thereby leading to the cap-independent translation of the gene. JacksonR J, Howell M T, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) andJackson R J and Kaminski, A. (1995) RNA 1(10):985-1000). The presentinvention encompasses the use of any IRES element which is able topromote direct internal ribosome entry to the initiation codon of acistron. “Under translational control of an IRES” as used herein meansthat translation is associated with the IRES and proceeds in acap-independent manner. Examples of “IRES” known in the art include, butare not limited to IRES obtainable from picornavirus (Jackson et al.,1990, Trends Biochem Sci 15(12):477-483); and IRES obtainable from viralor cellular mRNA sources, such as for example, immunogloublinheavy-chain binding protein (BiP), the vascular endothelial growthfactor (VEGF) (Huez et al. (1998) Mol. Cell. Biol. 18(11):6178-6190),the fibroblast growth factor 2, and insulin-like growth factor, thetranslational initiation factor eIF4G, yeast transcription factors TFIIDand HAP4. IRES have also been reported in different viruses such ascardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus(FrMLV) and Moloney murine leukemia virus (MoMLV). As used herein,“IRES” encompasses functional variations of IRES sequences as long asthe variation is able to promote direct internal ribosome entry to theinitiation codon of a cistron. In preferred embodiments, the IRES ismammalian. In other embodiments, the IRES is viral or protozoan. In oneillustrative embodiment disclosed herein, the IRES is obtainable fromencephelomycarditis virus (ECMV) (commercially available from Novogen,Duke et al. (1992) J. Virol 66(3):1602-1609). In another illustrativeembodiment disclosed herein, the IRES is from VEGF. Table I and Table IIdisclose a variety of IRES sequences useful in the present invention.

[0073] A “multicistronic transcript” refers to an mRNA molecule whichcontains more than one protein coding region, or cistron. A mRNAcomprising two coding regions is denoted a “bicistronic transcript.” The“5′-proximal” coding region or cistron is the coding region whosetranslation initiation codon (usually AUG) is closest to the 5′-end of amulticistronic mRNA molecule. A “5′-distal” coding region or cistron isone whose translation initiation codon (usually AUG) is not the closestinitiation codon to the 5′ end of the mRNA. The terms “5′-distal” and“downstream” are used synonymously to refer to coding regions that arenot adjacent to the 5′ end of a mRNA molecule.

[0074] As used herein, “co-transcribed” means that two (or more) codingregions of polynucleotides are under transcriptional control of singletranscriptional control element.

[0075] A “gene” refers to a coding region of a polynucleotide. A “gene”may or may not include non-coding sequences and/or regulatory elements.

[0076] As used herein, a “transcription response element” or“transcriptional regulatory element”, or “TRE” is a polynucleotidesequence, preferably a DNA sequence, which increases transcription of anoperably linked polynucleotide sequence in a host cell that allows thatTRE to function. A TRE can comprise an enhancer and/or a promoter. A“transcriptional regulatory sequence” is a TRE. A “target cell-specifictranscriptional response element” or “target cell-specific TRE” is apolynucleotide sequence, preferably a DNA sequence, which ispreferentially functional in a specific type of cell, that is, a targetcell. Accordingly, a target cell-specific TRE transcribes an operablylinked polynucleotide sequence in a target cell that allows the targetcell-specific TRE to function. The term “target cell-specific”, as usedherein, is intended to include cell type specificity, tissuespecificity, developmental stage specificity, and tumor specificity, aswell as specificity for a cancerous state of a given target cell.“Target cell-specific TRE” includes cell type-specific and cellstatus-specific TRE, as well as “composite” TREs. The term “compositeTRE” includes a TRE which comprises both a cell type-specific and a cellstatus-specific TRE. A target cell-specific TRE can also include aheterologous component, including, for example, an SV40 or acytomegalovirus (CMV) promoter(s). An example of a target cell specificTRE which is tissue specific is a CMV TRE which contains bothpromoter(s) and enhancer(s).

[0077] As described in more detail herein, a target cell-specific TREcan comprise any number of configurations, including, but not limitedto, a target cell-specific promoter; and target cell-specific enhancer;a heterologous promoter and a target cell-specific enhancer; a targetcell-specific promoter and a heterologous enhancer; a heterologouspromoter and a heterologous enhancer; and multimers of the foregoing.The promoter and enhancer components of a target cell-specific TRE maybe in any orientation and/or distance from the coding sequence ofinterest, as long as the desired target cell-specific transcriptionalactivity is obtained. Transcriptional activation can be measured in anumber of ways known in the art (and described in more detail below),but is generally measured by detection and/or quantitation of mRNA orthe protein product of the coding sequence under control of (i.e.,operably linked to) the target cell-specific TRE. As discussed herein, atarget cell-specific TRE can be of varying lengths, and of varyingsequence composition. As used herein, the term “cell status-specificTRE” is preferentially functional, i.e., confers transcriptionalactivation on an operably linked polynucleotide in a cell which allows acell status-specific TRE to function, i.e., a cell which exhibits aparticular physiological condition, including, but not limited to, anaberrant physiological state. “Cell status” thus refers to a given, orparticular, physiological state (or condition) of a cell, which isreversible and/or progressive. The physiological state may be generatedinternally or externally; for example, it may be a metabolic state (suchas in response to conditions of low oxygen), or it may be generated dueto heat or ionizing radiation. “Cell status” is distinct from a “celltype”, which relates to a differentiation state of a cell, which undernormal conditions is irreversible. Generally (but not necessarily), asdiscussed herein, a cell status is embodied in an aberrant physiologicalstate, examples of which are given below.

[0078] A “functional portion” of a target cell-specific TRE is one whichconfers target cell-specific transcription on an operably linked gene orcoding region, such that the operably linked gene or coding region ispreferentially expressed in the target cells.

[0079] By “transcriptional activation” or an “increase intranscription,” it is intended that transcription is increased abovebasal levels in the target cell (i.e., target cell) by at least about 2fold, preferably at least about 5 fold, preferably at least about 10fold, more preferably at least about 20 fold, more preferably at leastabout 50 fold, more preferably at least about 100 fold, more preferablyat least about 200 fold, even more preferably at least about 400 fold toabout 500 fold, even more preferably at least about 1000 fold. Basallevels are generally the level of activity (if any) in a non-target cell(i.e., a different cell type), or the level of activity (if any) of areporter construct lacking a target cell-specific TRE as tested in atarget cell line.

[0080] A “functionally-preserved variant” of a target cell-specific TREis a target cell-specific TRE which differs from another targetcell-specific TRE, but still retains target cell-specific transcriptionactivity, although the degree of activation may be altered (as discussedbelow). The difference in a target cell-specific TRE can be due todifferences in linear sequence, arising from, for example, single basemutation(s), addition(s), deletion(s), and/or modification(s) of thebases. The difference can also arise from changes in the sugar(s),and/or linkage(s) between the bases of a target cell-specific TRE. Forexample, certain point mutations within sequences of TREs have beenshown to decrease transcription factor binding and stimulation oftranscription. See Blackwood, et al. (1998) Science 281:60-63 and Smithet al. (1997) J. Biol. Chem. 272:27493-27496. One of skill in the artwould recognize that some alterations of bases in and aroundtranscription factor binding sites are more likely to negatively affectstimulation of transcription and cell-specificity, while alterations inbases which are not involved in transcription factor binding are not aslikely to have such effects. Certain mutations are also capable ofincreasing TRE activity. Testing of the effects of altering bases may beperformed in vitro or in vivo by any method known in the art, such asmobility shift assays, or transfecting vectors containing thesealterations in TRE functional and TRE non-functional cells.Additionally, one of skill in the art would recognize that pointmutations and deletions can be made to a TRE sequence without alteringthe ability of the sequence to regulate transcription.

[0081] As used herein, a TRE derived from a specific gene is referred toby the gene from which it was derived and is a polynucleotide sequencewhich regulates transcription of an operably linked polynucleotidesequence in a host cell that expresses said gene. For example, as usedherein, a “human glandular kallikrein transcriptional regulatoryelement”, or “hKLK2-TRE” is a polynucleotide sequence, preferably a DNAsequence, which increases transcription of an operably linkedpolynucleotide sequence in a host cell that allows an hKLK2-TRE tofunction, such as a cell (preferably a mammalian cell, even morepreferably a human cell) that expresses androgen receptor, such as aprostate cell. An hKLK2-TRE is thus responsive to the binding ofandrogen receptor and comprises at least a portion of an hKLK2 promoterand/or an hKLK2 enhancer (i.e., the ARE or androgen receptor bindingsite).

[0082] As used herein, a “probasin (PB) transcriptional regulatoryelement”, or “PB-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription of anoperably-linked polynucleotide sequence in a host cell that allows aPB-TRE to function, such as a cell (preferably a mammalian cell, morepreferably a human cell, even more preferably a prostate cell) thatexpresses androgen receptor. A PB-TRE is thus responsive to the bindingof androgen receptor and comprises at least a portion of a PB promoterand/or a PB enhancer (i.e., the ARE or androgen receptor binding site).

[0083] As used herein, a “prostate-specific antigen (PSA)transcriptional regulatory element”, or “PSA-TRE”, or “PSE-TRE” is apolynucleotide sequence, preferably a DNA sequence, which selectivelyincreases transcription of an operably linked polynucleotide sequence ina host cell that allows a PSA-TRE to function, such as a cell(preferably a mammalian cell, more preferably a human cell, even morepreferably a prostate cell) that expresses androgen receptor. A PSA-TREis thus responsive to the binding of androgen receptor and comprises atleast a portion of a PSA promoter and/or a PSA enhancer (i.e., the AREor androgen receptor binding site).

[0084] As used herein, a “carcinoembryonic antigen (CEA) transcriptionalregulatory element”, or “CEA-TRE” is a polynucleotide sequence,preferably a DNA sequence, which selectively increases transcription ofan operably linked polynucleotide sequence in a host cell that allows aCEA-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses CEA. The CEA-TRE isresponsive to transcription factors and/or co-factor(s) associated withCEA-producing cells and comprises at least a portion of the CEA promoterand/or enhancer.

[0085] As used herein, an “α-fetoprotein (AFP) transcriptionalregulatory element”, or “AFP-TRE” is a polynucleotide sequence,preferably a DNA sequence, which selectively increases transcription (ofan operably linked polynucleotide sequence) in a host cell that allowsan AFP-TRE to function, such as a cell (preferably a mammalian cell,even more preferably a human cell) that expresses AFP. The AFP-TRE isresponsive to transcription factors and/or co-factor(s) associated withAFP-producing cells and comprises at least a portion of the AFP promoterand/or enhancer.

[0086] As used herein, an “a mucin gene (MUC) transcriptional regulatoryelement”, or “MUC1-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription (of anoperably-linked polynucleotide sequence) in a host cell that allows aMUC1-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses MUC1. The MUC1-TRE isresponsive to transcription factors and/or co-factor(s) associated withMUC 1-producing cells and comprises at least a portion of the MUC1promoter and/or enhancer.

[0087] As used herein, a “urothelial cell-specific transcriptionalresponse element”, or “urothelial cell-specific TRE” is polynucleotidesequence, preferably a DNA sequence, which increases transcription of anoperably linked polynucleotide sequence in a host cell that allows aurothelial-specific TRE to function, i.e., a target cell. A variety ofurothelial cell-specific TREs are known, are responsive to cellularproteins (transcription factors and/or co-factor(s)) associated withurothelial cells, and comprise at least a portion of aurothelial-specific promoter and/or a urothelial-specific enhancer.Methods are described herein for measuring the activity of a urothelialcell-specific TRE and thus for determining whether a given cell allows aurothelial cell-specific TRE to function.

[0088] As used herein, a “melanocyte cell-specific transcriptionalresponse element”, or “melanocyte cell-specific TRE” is polynucleotidesequence, preferably a DNA sequence, which increases transcription of anoperably linked polynucleotide sequence in a host cell that allows amelanocyte-specific TRE to function, i.e., a target cell. A variety ofmelanocyte cell-specific TREs are known, are responsive to cellularproteins (transcription factors and/or co-factor(s)) associated withmelanocyte cells, and comprise at least a portion of amelanocyte-specific promoter and/or a melanocyte-specific enhancer.Methods are described herein for measuring the activity of a melanocytecell-specific TRE and thus for determining whether a given cell allows amelanocyte cell-specific TRE to function.

[0089] An “E1B 19-kDa region” (used interchangeably with “E1B 19-kDagenomic region”) refers to the genomic region of the adenovirus E1B geneencoding the E1B 19-kDa product. According to wild-type Ad5, the E1B19-kDa region is a 261 bp region located between nucleotide 1714 andnucleotide 2244. The E1B 19-kDa region has been described in, forexample, Rao et al., Proc. Natl. Acad. Sci. USA, 89:7742-7746. Thepresent invention encompasses deletion of part or all of the E1B 19-kDaregion as well as embodiments wherein the E1B 19-kDa region is mutated,as long as the deletion or mutation lessens or eliminates the inhibitionof apoptosis associated with E1B-19 kDa.

[0090] As used herein, a target cell-specific TRE can comprise anynumber of configurations, including, but not limited to, a targetcell-specific promoter; a target cell-specific enhancer; a targetcell-specific promoter and a target cell-specific enhancer; a targetcell-specific promoter and a heterologous enhancer; a heterologouspromoter and a target cell-specific enhancer; and multimers of theforegoing. The promoter and enhancer components of a targetcell-specific TRE may be in any orientation and/or distance from thecoding sequence of interest, as long as the desired target cell-specifictranscriptional activity is obtained. Transcriptional activation can bemeasured in a number of ways known in the art (and described in moredetail below), but is generally measured by detection and/orquantitation of mRNA or the protein product of the coding sequence undercontrol of (i.e., operably linked to) the target cell-specific TRE.

[0091] “Replicating preferentially”, as used herein, means that theadenovirus replicates more in a target cell than a non-target cell.Preferably, the adenovirus replicates at a significantly higher rate intarget cells than non target cells; preferably, at least about 2-foldhigher, preferably, at least about 5-fold higher, more preferably, atleast about 10-fold higher, still more preferably at least about 50-foldhigher, even more preferably at least about 100-fold higher, still morepreferably at least about 400- to 500-fold higher, still more preferablyat least about 1000-fold higher, most preferably at least about 1×10⁶higher. Most preferably, the adenovirus replicates solely in the targetcells (that is, does not replicate or replicates at a very low levels innon-target cells).

[0092] As used herein, the term “vector” refers to a polynucleotideconstruct designed for transduction/transfection of one or more celltypes. Vectors may be, for example, “cloning vectors” which are designedfor isolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector.

[0093] An “adenovirus vector” or “adenoviral vector” (usedinterchangeably) comprises a polynucleotide construct of the invention.A polynucleotide construct of this invention may be in any of severalforms, including, but not limited to, DNA, DNA encapsulated in anadenovirus coat, DNA packaged in another viral or viral-like form (suchas herpes simplex, and AAV), DNA encapsulated in liposomes, DNAcomplexed with polylysine, complexed with synthetic polycationicmolecules, conjugated with transferrin, and complexed with compoundssuch as PEG to immunologically “mask” the molecule and/or increasehalf-life, and conjugated to a nonviral protein. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides. For purposes of this invention, adenovirus vectors arereplication-competent in a target cell.

[0094] The terms “polynucleotide” and “nucleic acid”, usedinterchangeably herein, refer to a polymeric form of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides. These termsinclude a single-, double- or triple-stranded DNA, genomic DNA, cDNA,RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically, biochemically modified, non-naturalor derivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the polynucleotide can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be aoligodeoxynucleoside phosphoramidate (P-NH2) or a mixedphosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) NucleicAcids Res. 24: 1841-8; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-23; Schultz et al. (1996) Nucleic Acids Res. 24: 2966-73. Aphosphorothioate linkage can be used in place of a phosphodiesterlinkage. Braun et al. (1988) J. Immunol. 141: 2084-9; Latimer et al.(1995) Molec. Immunol. 32: 1057-1064. In addition, a double-strandedpolynucleotide can be obtained from the single stranded polynucleotideproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands under appropriate conditions, or bysynthesizing the complementary strand de novo using a DNA polymerasewith an appropriate primer. Reference to a polynucleotide sequence (suchas referring to a SEQ ID NO) also includes the complement sequence.

[0095] The following are non-limiting examples of polynucleotides: agene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

[0096] A polynucleotide or polynucleotide region has a certainpercentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity”to another sequence means that, when aligned, that percentage of basesare the same in comparing the two sequences. This alignment and thepercent homology or sequence identity can be determined using softwareprograms known in the art, for example those described in CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987)Supplement 30, section 7.7.18. A preferred alignment program is ALIGNPlus (Scientific and Educational Software, Pennsylvania), preferablyusing default parameters, which are as follows: mismatch=2; open gap=0;extend gap=2.

[0097] “Under transcriptional control” is a term well understood in theart and indicates that transcription of a polynucleotide sequence,usually a DNA sequence, depends on its being operably (operatively)linked to an element which contributes to the initiation of, orpromotes, transcription. “Operably linked” refers to a juxtapositionwherein the elements are in an arrangement allowing them to function.

[0098] An “E3 region” (used interchangeably with “E3”) is a term wellunderstood in the art and means the region of the adenoviral genome thatencodes the E3 products (discussed herein). Generally, the E3 region islocated between about 28583 and 30470 of the adenoviral genome. The E3region has been described in various publications, including, forexample, Wold et al. (1995) Curr. Topics Microbiol. Immunol.199:237-274.

[0099] A “portion” of the E3 region means less than the entire E3region, and as such includes polynucleotide deletions as well aspolynucleotides encoding one or more polypeptide products of the E3region.

[0100] As used herein, “cytotoxicity” is a term well understood in theart and refers to a state in which a cell's usual biochemical orbiological activities are compromised (i.e., inhibited). Theseactivities include, but are not limited to, metabolism; cellularreplication; DNA replication; transcription; translation; uptake ofmolecules. “Cytotoxicity” includes cell death and/or cytolysis. Assaysare known in the art which indicate cytotoxicity, such as dye exclusion,³H-thymidine uptake, and plaque assays.

[0101] The term “selective cytotoxicity”, as used herein, refers to thecytotoxicity conferred by an adenovirus vector of the present inventionon a cell which allows or induces a target cell-specific TRE to function(a target cell) when compared to the cytotoxicity conferred by anadenoviral vector of the present invention on a cell which does notallow a target cell-specific TRE to function (a non-target cell). Suchcytotoxicity may be measured, for example, by plaque assays, byreduction or stabilization in size of a tumor comprising target cells,or the reduction or stabilization of serum levels of a markercharacteristic of the tumor cells, or a tissue-specific marker, e.g., acancer marker.

[0102] In the context of adenovirus, a “heterologous polynucleotide” or“heterologous gene” or “transgene” is any polynucleotide or gene that isnot present in wild-type adenovirus. Preferably, the transgene will alsonot be expressed or present in the target cell prior to introduction bythe adenovirus vector. Examples of preferred transgenes are providedbelow.

[0103] In the context of adenovirus, a “heterologous” promoter orenhancer is one which is not associated with or derived from anadenovirus gene.

[0104] In the context of adenovirus, an “endogenous” promoter, enhancer,or TRE is native to or derived from adenovirus. In the context ofpromoter, an “inactivation” means that there is a mutation of ordeletion in part or all of the of the endogenous promoter, ie, amodification or alteration of the endogenous promoter, such as, forexample, a point mutation or insertion, which disables the function ofthe promoter.

[0105] In the context of a target cell-specific TRE, a “heterologous”promoter or enhancer is one which is derived from a gene other than thegene from which a reference target cell-specific TRE is derived.

[0106] “Suppressing” tumor growth indicates a growth state that iscurtailed when compared to growth without contact with, i.e.,transfection by, an adenoviral vector described herein. Tumor cellgrowth can be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and stopping tumor growth, as wellas tumor shrinkage.

[0107] As used herein, the terms “neoplastic cells”, “neoplasia”,“tumor”, “tumor cells”, “cancer” and “cancer cells”, (usedinterchangeably) refer to cells which exhibit relatively autonomousgrowth, so that they exhibit an aberrant growth phenotype characterizedby a significant loss of control of cell proliferation (i.e.,de-regulated cell division). Neoplastic cells can be malignant orbenign.

[0108] A “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of an adenoviral vector(s) of thisinvention. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or intotal DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation and/or change. A host cell includescells transfected or infected in vivo or in vitro with an adenoviralvector of this invention.

[0109] “Replication” and “propagation” are used interchangeably andrefer to the ability of an adenovirus vector of the invention toreproduce or proliferate. These terms are well understood in the art.For purposes of this invention, replication involves production ofadenovirus proteins and is generally directed to reproduction ofadenovirus. Replication can be measured using assays standard in the artand described herein, such as a burst assay or plaque assay.“Replication” and “propagation” include any activity directly orindirectly involved in the process of virus manufacture, including, butnot limited to, viral gene expression; production of viral proteins,nucleic acids or other components; packaging of viral components intocomplete viruses; and cell lysis.

[0110] An “ADP coding sequence” is a polynucleotide that encodes ADP ora functional fragment thereof. In the context of ADP, a “functionalfragment” of ADP is one that exhibits cytotoxic activity, especiallycell lysis, with respect to adenoviral replication. Ways to measurecytotoxic activity are known in the art and are described herein.

[0111] A polynucleotide that “encodes” an ADP polypeptide is one thatcan be transcribed and/or translated to produce an ADP polypeptide or afragment thereof. The anti-sense strand of such a polynucleotide is alsosaid to encode the sequence.

[0112] An “ADP polypeptide” is a polypeptide containing at least aportion, or region, of the amino acid sequence of an ADP and whichdisplays a function associated with ADP, particularly cytotoxicity, moreparticularly, cell lysis. As discussed herein, these functions can bemeasured using techniques known in the art. It is understood thatcertain sequence variations may be used, due to, for example,conservative amino acid substitutions, which may provide ADPpolypeptides.

[0113] “Androgen receptor,” or AR, as used herein refers to a proteinwhose function is to specifically bind to androgen and, as a consequenceof the specific binding, recognize and bind to an androgen responseelement (ARE), following which the AR is capable of regulatingtranscriptional activity. The AR is a nuclear receptor that, whenactivated, binds to cellular androgen-responsive element(s). In normalcells the AR is activated by androgen, but in non-normal cells(including malignant cells) the AR may be activated by non-androgenicagents, including hormones other than androgens. Encompassed in the term“androgen receptor” are mutant forms of an androgen receptor, such asthose characterized by amino acid additions, insertions, truncations anddeletions, as long as the function is sufficiently preserved. Mutantsinclude androgen receptors with amino acid additions, insertions,truncations and deletions, as long as the function is sufficientlypreserved. In this context, a functional androgen receptor is one thatbinds both androgen and, upon androgen binding, an ARE.

[0114] A polynucleotide sequence that is “depicted in” a SEQ ID NO meansthat the sequence is present as an identical contiguous sequence in theSEQ ID NO. The term encompasses portions, or regions of the SEQ ID NO aswell as the entire sequence contained within the SEQ ID NO.

[0115] A “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

[0116] An “individual” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, farmanimals, sport animals, rodents, primates, and pets.

[0117] An “effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of an adenoviral vector is anamount that is sufficient to palliate, ameliorate, stabilize, reverse,slow or delay the progression of the disease state.

[0118] A given TRE is “derived from” a given gene if it is associatedwith that gene in nature.

[0119] “Expression” includes transcription and/or translation.

[0120] As used herein, the term “comprising” and its cognates are usedin their inclusive sense; that is, equivalent to the term “including”and its corresponding cognates.

[0121] “A,” “an” and “the” include plural references unless the contextclearly dictates otherwise.

[0122] Internal Ribosome Entry Site (IRES)

[0123] IRES elements were first discovered in picornavirus mRNAs(Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci15(12):477-83) and Jackson R J and Kaminski, A. (1995) RNA1(10):985-1000). The present invention provides improved adenovirusvectors comprising co-transcribed first and second genes undertranscriptional control of a heterologous, target cell-specific TRE, andwherein the second gene (i.e., coding region) is under translationalcontrol of an internal ribosome entry site (IRES). Any IRES may be usedin the adenovirus vectors of the invention, as long as they exhibitrequisite function in the vectors. Example of IRES which can be used inthe present invention include those provided in Table I and referencedin Table II. Examples of IRES elements include the encephelomycarditisvirus (EMCV) which is commercially available from Novagen (Duke et al.(1992) J. Virol 66(3):1602-9) the sequence for which is depicted inTable 1 (SEQ ID NO:1). Another example of an IRES element disclosedherein is the VEGF IRES (Huez et al. (1998) Mol Cell Biol18(11):6178-90). This IRES has a short segment and the sequence isdepicted in Table 1 (SEQ ID NO:2).

[0124] The IRES promotes direct internal ribosome entry to theinitiation codon of a downstream cistron, leading to cap-independenttranslation. Thus, the product of a downstream cistron can be expressedfrom a bicistronic (or multicistronic) mRNA, without requiring eithercleavage of a polyprotein or generation of a monocistronic mRNA.Therefore, in one illustrative embodiment of the present invention, anadenovirus vector comprising E1B under translational control of an IRESallows translation of E1B from a bicistronic E1A-E1B mRNA under controlof a target cell-specific TRE. FIG. 7 provides a schematicrepresentation of adenovirus constructs of the present invention.

[0125] Internal ribosome entry sites are approximately 450 nucleotidesin length and are characterized by moderate conservation of primarysequence and strong conservation of secondary structure. The mostsignificant primary sequence feature of the IRES is a pyrimidine-richsite whose start is located approximately 25 nucleotides upstream of the3′ end of the IRES. See Jackson et al. (1990).

[0126] Three major classes of picornavirus IRES have been identified andcharacterized: (1) the cardio- and aphthovirus class (for example, theencephelomycarditis virus, Jang et al. (1990) Gene Dev 4:1560-1572); (2)the entero- and rhinovirus class (for example, polioviruses, Borman etal. (1994) EMBO J. 13:314903157); and (3) the hepatitis A virus (HAV)class, Glass et al. (1993) Virol 193:842-852). For the first twoclasses, two general principles apply. First, most of the 450-nucleotidesequence of the IRES functions to maintain particular secondary andtertiary structures conducive to ribosome binding and translationalinitiation. Second, the ribosome entry site is an AUG triplet located atthe 3′ end of the IRES, approximately 25 nucleotides downstream of aconserved oligopyrimidine tract. Translation initiation can occur eitherat the ribosome entry site (cardioviruses) or at the next downstream AUG(entero/rhinovirus class). Initiation occurs at both sites inaphthoviruses.

[0127] HCV and pestiviruses such as bovine viral diarrhea virus (BVDV)or classical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTRrespectively. These 5′-UTR fragments form similar RNA secondarystructures and can have moderately efficient IRES function(Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-1483; Frolov I et al.,(1998) RNA 4:1418-1435). Table I depicts the 5′-UTR region from HCVgenome sequence (GenBank accession D14853).

[0128] Leishmania RNA virus 1 (LRV1) is a double-stranded RNA virus. Its128 nt long 5′-UTR has IRES activity to facilitate the cap-independenttranslation, (Maga et al. (1995) Mol Cell Biol 15:4884-4889). Thisfragment also forms conserved stemloop secondary structure and at leastthe front part is essential.

[0129] Recent studies showed that both Friend-murine leukemia virus(MLV) 5′-UTR and rat retrotransposon virus-like 30S (VL30) sequencescontain IRES structure of retroviral origin (Torrent et al. (1996) HumGene Ther 7:603-612). These fragments are also functional as packingsignal when used in retrovirus derived vectors. Studies of avianreticuloendotheliosis virus type A (REV-A) show that its IRES mapsdownstream of the packaging/dimerization (E/DLS) sequence and theminimal IRES sequence appears to be within a 129 nt fragment (452-580)of the 5′ leader, immediately upstream of the gag AUG codon(Lopez-Lastra et al. (1997) Hum Gene Ther 8:1855-1865).

[0130] In eukaryotic cells, translation is normally initiated by theribosome scanning from the capped mRNA 5′ end, under the control ofinitiation factors. However, several cellular mRNAs have been found tohave IRES structure to mediate the cap-independent translation (van derVelde, et al. (1999) Int J Biochem Cell Biol. 31:87-106). Examples areimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94), antennapedia mRNA of Drosophilan (Oh et al. (1992)Gene and Dev 6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner etal. (1995) Mol Cell Biol 15:35-44), platelet-derived growth factor B(PDGF-B) (Bernstein et al. (1997) J Biol Chem 272:9356-9362),insulin-like growth factor II (Teerink et al. (1995) Biochim BiophysActa 1264:403-408), and the translation initiation factor eIF4G (Gan etal. (1996) J Biol Chem 271:623-626). Table 1 depicts the 5′-noncodingregion for BiP and PDGF. Recently, vascular endothelial growth factor(VEGF) was also found to have IRES element (Stein et al. (1998) Mol CellBiol 18:3112-3119; Huez et al. (1998) Mol Cell Biol 18:6178-6190).

[0131] Apart from the oligopyrimidine tract, nucleotide sequence per sedoes not appear to be important for IRES function. Without wishing to bebound by theory, a possible explanation for the function of an IRES isthat it forms secondary and/or tertiary structures which orientparticular single-stranded regions of its sequence in athree-dimensional configuration that is conducive to interaction with amammalian ribosome (either ribosomal protein and/or ribosomal RNAcomponents) and/or initiation factor(s) and/or RNA binding proteinswhich interact with ribosomes and/or initiation factors. It is alsopossible that the three-dimensional structure of the IRES is determinedor stabilized by one or more RNA-binding proteins. Thus it is possibleto devise synthetic IRES sequences having similar single-strandedregions in a similar three-dimensional configuration.

[0132] In certain cases, one or more trans-acting cellular proteins maybe required for IRES function. For example, the HAV andentero/rhinovirus IRESes function inefficiently in vitro in reticulocytelysates. Supplementation of a reticulocyte lysate with a cytoplasmicextract from HeLa, Krebs II ascites, or L-cells restores activity ofentero/rhinovirus IRESes. See, for example, Brown et al. (1979) Virology97:396-405; and Dorner et al. (1984) J. Virol. 50:507-514. Activity ofthe HAV IRES in vitro is stimulated by liver cytoplasmic extracts. Glasset al. (1993) Virology 193:1047-1050. These observations indicate thatcell-specific translational regulation can be achieved through the useof a cell-specific IRES. Furthermore, coordinated cell-specifictranscriptional and translational regulatory elements can be included ina vector to further increase cell specificity of viral replication. Forexample, the combination of an AFP-TRE and a HAV-IRES can be used todirect preferential replication of a vector in hepatic cells. Thus, inone illustrative embodiment, a vector comprises an AFP-TRE regulatingthe transcription of a bicistronic E1A-E1B mRNA in which E1B translationis regulated by an ECMV IRES. In another illustrative embodiment, thevector comprises a probasin-TRE regulating the transcription of abicistronic E1A-E1B mRNA in which E1B translation is regulated by anECMV IRES. In yet another illustrative embodiment, a vector comprises aCMV-TRE regulating the transcription of a bicistronic E1A-E1B mRNA inwhich E1B translation is regulated by an ECMV IRES.

[0133] Examples of IRES which can be used in the present inventioninclude those provided in Table 1 and Table 2. An IRES sequence whichmay be used in the present invention may be tested as follows. A testvector is produced having a reporter gene, such as luciferase, forexample, placed under translational control of an IRES to be tested. Adesired cell type is transfected with the vector containing the desiredIRES-reporter gene and an assay is performed to detect the presence ofthe reporter gene. In one illustrative example, the test vectorcomprises a co-transcribed chloramphenicol transferase (CAT) andluciferase encoding gene transcriptionally driven by a CMV promoterwherein the luciferase encoding gene is translationally driven by anIRES to be tested. Host cells are transiently transfected with the testvector by means known to those of skill in the art and assayed for thepresence of luciferase.

[0134] IRES may be prepared using standard recombinant and syntheticmethods known in the art, and as described in the Examples. For cloningconvenience, restriction sites may be engineered into the ends of theIRES fragments to be used.

[0135] Transcriptional Response Elements (TREs)

[0136] The adenovirus vectors of the invention comprise target cellspecific TREs which direct preferential expression of an operativelylinked gene (or genes) in a particular target cell. A TRE can betissue-specific, tumor-specific, developmental stage-specific, cellstatus specific, etc., depending on the type of cell present in thetissue or tumor.

[0137] Cell- and tissue-specific transcriptional regulatory elements, aswell as methods for their identification, isolation, characterization,genetic manipulation and use for regulation of operatively linked codingsequences, are well known in the art. A TRE can be derived from thetranscriptional regulatory sequences of a single gene, or sequences fromdifferent genes can be combined to produce a functional TRE. Acell-specific TRE is preferentially functional in a limited population(or type) of cells, e.g., prostate cells or liver cells. Accordingly, insome embodiments, the TRE used is preferentially functional in any ofthe following cell types: prostate; liver; breast; urothelial cells(bladder); colon; lung; ovarian; pancreas; stomach; and uterine. Inother embodiments, in accordance with cell status, the TRE is functionalin or during: low oxygen conditions (hypoxia); certain stages of cellcycle, such as S phase; elevated temperature; ionizing radiation.

[0138] As is known in the art, activity of TREs can be inducible.Inducible TREs generally exhibit low activity in the absence of inducer,and are up-regulated in the presence of inducer. Inducers include, forexample, nucleic acids, polypeptides, small molecules, organic compoundsand/or environmental conditions such as temperature, pressure orhypoxia. Inducible TREs may be preferred when expression is desired onlyat certain times or at certain locations, or when it is desirable totitrate the level of expression using an inducing agent. For example,transcriptional activity from the PSE-TRE, PB-TRE and hKLK2-TRE isinducible by androgen, as described herein and in PCT/US98/04080.Accordingly, in one embodiment of the present invention, an adenovirusvector comprises an inducible heterologous TRE.

[0139] TRE multimers are also useful in the disclosed vectors. Forexample, a TRE can comprise a tandem series of at least two, at leastthree, at least four, or at least five promoter fragments.Alternatively, a TRE can comprise one or more promoter regions alongwith one or more enhancer regions. TRE multimers can also comprisepromoter and/or enhancer sequences from different genes. The promoterand enhancer components of a TRE can be in any orientation with respectto each other and can be in any orientation and/or any distance from thecoding sequence of interest, as long as the desired cell-specifictranscriptional activity is obtained.

[0140] The disclosed vectors are designed such that replication ispreferentially enhanced in target cells in which the TRE(s) is (are)functional. More than one TRE can be present in a vector, as long as theTREs are functional in the same target cell. However, it is important tonote that a given TRE can be functional in more than one type of targetcell. For example, the CEA-TRE functions in, among other cell types,gastric cancer cells, colorectal cancer cells, pancreatic cancer cellsand lung cancer cells.

[0141] A TRE for use in the present vectors may or may not comprise asilencer. The presence of a silencer (i.e., a negative regulatoryelement known in the art) can assist in shutting off transcription (andthus replication) in non-target cells. Thus, presence of a silencer canconfer enhanced cell-specific vector replication by more effectivelypreventing replication in non-target cells. Alternatively, lack of asilencer may stimulate replication in target cells, thus conferringenhanced target cell-specificity.

[0142] As is readily appreciated by one skilled in the art, a TRE is apolynucleotide sequence, and, as such, can exhibit function over avariety of sequence permutations. Methods of nucleotide substitution,addition, and deletion are known in the art, and readily-availablefunctional assays (such as the CAT or luciferase reporter gene assay)allow one of ordinary skill to determine whether a sequence variantexhibits requisite cell-specific transcription regulatory function.Hence, functionally preserved variants of TREs, comprising nucleic acidsubstitutions, additions, and/or deletions, can be used in the vectorsdisclosed herein. Accordingly, variant TREs retain function in thetarget cell but need not exhibit maximal function. In fact, maximaltranscriptional activation activity of a TRE may not always be necessaryto achieve a desired result, and the level of induction afforded by afragment of a TRE may be sufficient for certain applications. Forexample, if used for treatment or palliation of a disease state,less-than-maximal responsiveness may be sufficient if, for example, thetarget cells are not especially virulent and/or the extent of disease isrelatively confined.

[0143] Certain base modifications may result in enhanced expressionlevels and/or cell-specificity. For example, nucleic acid sequencedeletions or additions within a TRE can move transcription regulatoryprotein binding sites closer or farther away from each other than theyexist in their normal configuration, or rotate them so they are onopposite sides of the DNA helix, thereby altering spatial relationshipamong TRE-bound transcription factors, resulting in a decrease orincrease in transcription, as is known in the art. Thus, while notwishing to be bound by theory, the present disclosure contemplates thepossibility that certain modifications of a TRE will result in modulatedexpression levels as directed by the TRE, including enhancedcell-specificity. Achievement of enhanced expression levels may beespecially desirable in the case of more aggressive forms of neoplasticgrowth, and/or when a more rapid and/or aggressive pattern of cellkilling is warranted (for example, in an immunocompromised individual).

[0144] Transcriptional activity directed by a TRE (including bothinhibition and enhancement) can be measured in a number of ways known inthe art (and described in more detail below), but is generally measuredby detection and/or quantitation of mRNA and/or of a protein productencoded by the sequence under control of (i.e., operably linked to) aTRE.

[0145] As discussed herein, a TRE can be of varying lengths, and ofvarying sequence composition. The size of a heterologous TRE will bedetermined in part by the capacity of the viral vector, which in turndepends upon the contemplated form of the vector (see infra). Generallyminimal sizes are preferred for TREs, as this provides potential roomfor insertion of other sequences which may be desirable, such astransgenes (discussed infra) and/or additional regulatory sequences. Ina preferred embodiment, such an additional regulatory sequence is anIRES. However, if no additional sequences are contemplated, or if, forexample, an adenoviral vector will be maintained and delivered free ofany viral packaging constraints, larger TRE sequences can be used aslong as the resultant adenoviral vector remains replication-competent.

[0146] An adenoviral vector can be packaged with extra sequencestotaling up to about 5% of the genome size, or approximately 1.8 kb,without requiring deletion of viral sequences. If non-essentialsequences are removed from the adenovirus genome, an additional 4.6 kbof insert can be tolerated (i.e., for a total insertion capacity ofabout 6.4 kb). Examples of non-essential adenoviral sequences that canbe deleted are E3, and E4 sequences other than those which encode E4ORF6.

[0147] To minimize non-specific replication, endogenous (e.g.,adenovirus) TREs are preferably removed from the vector. Besidesfacilitating target cell-specific replication, removal of endogenousTREs also provides greater insert capacity in a vector, which may be ofspecial concern if an adenoviral vector is to be packaged within a virusparticle. Even more importantly, deletion of endogenous TREs preventsthe possibility of a recombination event whereby a heterologous TRE isdeleted and the endogenous TRE assumes transcriptional control of itsrespective adenovirus coding sequences (thus allowing non-specificreplication). In one embodiment, an adenoviral vector is constructedsuch that the endogenous transcription control sequences of adenoviralgenes are deleted and replaced by one or more heterologous TREs.However, endogenous TREs can be maintained in the adenovirus vector(s),provided that sufficient cell-specific replication preference ispreserved. These embodiments are constructed by inserting heterologousTREs between an endogenous TRE and a replication gene coding segment.Requisite cell-specific replication preference is determined byconducting assays that compare replication of the adenovirus vector in acell which allows function of the heterologous TREs with replication ina cell which does not.

[0148] Generally, a TRE will increase replication of a vector in atarget cell by at least about 2-fold, preferably at least about 5-fold,preferably at least about 10-fold more preferably at least about20-fold, more preferably at least about 50-fold, more preferably atleast about 100-fold, more preferably at least about 200-fold, even morepreferably at least about 400- to about 500-fold, even more preferablyat least about 1000-fold, compared to basal levels of replication in theabsence of a TRE. The acceptable differential can be determinedempirically (by measurement of mRNA levels using, for example, RNA blotassays, RNase protection assays or other assays known in the art) andwill depend upon the anticipated use of the vector and/or the desiredresult.

[0149] Replication-competent adenovirus vectors directed at specifictarget cells can be generated using TREs that are preferentiallyfunctional in a target cell. In one embodiment of the present invention,the target cell is a tumor cell. Non-limiting examples of tumorcell-specific heterologous TREs, and their respective target cells,include: probasin (PB), target cell, prostate cancer (PCT/US98/04132);α-fetoprotein (AFP), target cell liver cancer (PCT/US98/04084);mucin-like glycoprotein DF3 (MUC1), target cell, breast carcinoma(PCT/US98/04080); carcinoembryonic antigen (CEA), target cells,colorectal, gastric, pancreatic, breast, and lung cancers(PCT/US98/04133); plasminogen activator urokinase (uPA) and its receptorgene, target cells, breast, colon, and liver cancers (PCT/US98/04080);E2F] (cell cycle S-phase specific promoter); target cell, tumors withdisrupted retinoblastoma gene function, and HER-2/neu (c-erbB2/neu),target cell, breast, ovarian, stomach, and lung cancers(PCT/US98/04080); tyrosinase, target cell, melanoma cells as describedherein and uroplakins, target cell, bladder cells as described herein.Methods for identification, isolation, characterization and utilizationof additional target cell-specific TREs are readily available to thoseof skill in the art.

[0150] In addition, tumor-specific TREs can be used in conjunction withtissue-specific TREs from the following exemplary genes (tissue in whichthe TREs are specifically functional are in parentheses): hypoxiaresponsive element, vascular endothelial growth factor receptor(endothelium), albumin (liver), factor VII (liver), fatty acid synthase(liver), Von Willebrand factor (brain endothelium), alpha-actin andmyosin heavy chain (both in smooth muscle), synthetase I (smallintestine) Na⁺—K⁺—Cl⁻ transporter (kidney). Additional tissue-specificTREs are known in the art.

[0151] In one embodiment of the present invention, a targetcell-specific, heterologous TRE is tumor cell-specific. A vector cancomprise a single tumor cell-specific TRE or multiple heterologous TREswhich are tumor cell-specific and functional in the same cell. Inanother embodiment, a vector comprises one or more heterologous TREswhich are tumor cell-specific and additionally comprises one or moreheterologous TREs which are tissue specific, whereby all TREs arefunctional in the same cell.

[0152] Prostate-Specific TREs

[0153] In one embodiment, adenovirus vectors comprise heterologous TREsthat are prostate cell specific. For example, TREs that functionpreferentially in prostate cells and can be used to target adenovirusreplication to prostate neoplasia, include, but are not limited to, TREsderived from the prostate-specific antigen gene (PSA-TRE) (HendersonU.S. Pat. No. 5,698,443); the glandular kallikrein-1 gene (from thehuman gene, hKLK2-TRE) (PCT US98/16312), and the probasin gene (PB-TRE)(PCT[US98/04132). All three of these genes are preferentially expressedin prostate cells and their expression is androgen-inducible. Generally,expression of genes responsive to androgen induction is mediated by anandrogen receptor (AR).

[0154] Prostate-Specific Antigen (PSA)

[0155] PSA is synthesized exclusively in prostatic epithelial cells andis synthesized in these cells whether they are normal, hyperplastic, ormalignant. This tissue-specific expression of PSA has made it anexcellent biomarker for benign prostatic hyperplasia (BPH) and prostaticcarcinoma (CaP). Normal serum levels of PSA are typically below 5 ng/ml,with elevated levels indicative of BPH or CaP. Lundwall et al. (1987)FEBS Lett. 214:317; Lundwall (1989) Biochem. Biophys. Res. Comm.161:1151; and Riegmann et al. (1991) Molec. Endocrin. 5:1921.

[0156] The region of the PSA gene that provides androgen-dependent cellspecificity, particularly in prostate cells, involves approximately 6.0kilobases (kb). Schuur et al. (1996) J. Biol. Chem. 271:7043-7051. Anenhancer region of approximately 1.5 kb in humans is located between nt−5322 and nt −3739, relative to the transcription start site of the PSAgene. Within these enhancer sequences is an androgen response element(ARE) a sequence which binds androgen receptor. The sequence coordinatesof the PSA promoter are from about nt −540 to nt+8 relative to thetranscription start site. Juxtapositioning of the enhancer and promoteryields a fully functional, minimal prostate-specific TRE (PSA-TRE).Other portions of this approximately 6.0 kb region of the PSA gene canbe used in the vectors described herein, as long as requisitefunctionality is maintained.

[0157] Human Glandular Kallikrein (hKLK2)

[0158] Human glandular kallikrein (hKLK2, encoding the hK2 protein) isexpressed exclusively in the prostate and its expression is up-regulatedby androgens, primarily through transcriptional activation. Wolf et al.(1992) Molec. Endocrinol. 6:753-762; Morris (1989) Clin. Exp. Pharm.Physiol. 16:345-351; Qui et al. (1990) J. Urol. 144:1550-1556; and Younget al. (1992) Biochem. 31:818-824. The levels of hK2 found in varioustumors and in the serum of patients with prostate cancer indicate thathK2 antigen may be a significant marker for prostate cancer.Charlesworth et al. (1997) Urology 49:487-493. Expression of hK2 hasbeen detected in each of 257 radical prostatectomy specimens analyzed.Darson et al. (1997) Urology 49:857-862. The intensity and extent of hK2expression, detected using specific antibodies, was observed to increasefrom benign epithelium to high-grade prostatic intraepithelial neoplasia(PIN) and adenocarcinoma.

[0159] The activity of the hKLK2 promoter has been described and aregion up to nt −2256 relative to the transcription start site waspreviously disclosed. Schedlich et al. (1987) DNA 6:429-437. The hKLK2promoter is androgen responsive and, in plasmid constructs wherein thepromoter alone controls the expression of a reporter gene, expression ofthe reporter gene is increased approximately 10-fold in the presence ofandrogen. Murtha et al. (1993) Biochem. 32:6459-6464. hKLK2 enhanceractivity is found within a polynucleotide sequence approximately nt−12,014 to nt −2257 relative to the start of transcription and, whenthis sequence is operably linked to an hKLK2 promoter and a reportergene, transcription of operably-linked sequences in prostate cellsincreases in the presence of androgen to levels approximately 30-fold toapproximately 100-fold greater than the level of transcription in theabsence of androgen. This induction is generally independent of theorientation and position of the enhancer sequences. Enhancer activityhas also been demonstrated in the following regions (all relative to thetranscription start site): about nt −3993 to about nt −3643, about nt−4814 to about nt −3643, about nt −5155 to about nt −3387, about nt−6038 to about nt −2394.

[0160] Thus, a hKLK2 enhancer can be operably linked to an hKLK2promoter or a heterologous promoter to form a hKLK2 transcriptionalregulatory element (hKLK2-TRE). A hKLK2-TRE can then be operably linkedto a heterologous polynucleotide to confer hKLK2-TRE-specifictranscriptional regulation on the linked gene, thus increasing itsexpression.

[0161] Probasin

[0162] The rat probasin (PB) gene encodes an androgen and zinc-regulatedprotein first characterized in the dorsolateral prostate of the rat.Dodd et al. (1983) J. Biol. Chem. 258:10731-10737; Matusik et al. (1986)Biochem. Cell. Biol. 64:601-607; and Sweetland et al. (1988) Mol. Cell.Biochem. 84:3-15. The dorsolateral lobes of the murine prostate areconsidered the most homologous to the peripheral zone of the humanprostate, where approximately 68% of human prostate cancers are thoughtto originate.

[0163] A PB-TRE has been shown to exist in an approximately 0.5 kbfragment of sequence upstream of the probasin coding sequence, fromabout nt −426 to about nt+28 relative to the transcription start site.This minimal promoter sequence from the PB gene appears to providesufficient information to direct prostate-specific developmental- andhormone-regulated expression of an operably linked heterologous gene intransgenic mice. Greenberg et al. (1994) Mol. Endocrinol. 8:230-239.

[0164] Alpha-Fetoprotein

[0165] α-fetoprotein (AFP) is an oncofetal protein, the expression ofwhich is primarily restricted to developing tissues of endodermal origin(yolk sac, fetal liver, and gut), although the level of its expressionvaries greatly depending on the tissue and the developmental stage. AFPis of clinical interest because the serum concentration of AFP iselevated in a majority of hepatoma patients, with high levels of AFPfound in patients with advanced disease. High serum AFP levels inpatients appear to be due to AFP expression in hepatocellular carcinoma(HCC), but not in surrounding normal liver. Thus, expression of the AFPgene appears to be characteristic of hepatoma cells. An AFP-TRE isdescribed in for example PCT/US98/04084.

[0166] According to published reports, the AFP-TRE is responsive tocellular proteins (transcription factors and/or co-factor(s)) associatedwith AFP-producing cells, such as AFP-binding protein (see, for example,U.S. Pat. No. 5,302,698) and comprises at least a portion of an AFPpromoter and/or an AFP enhancer. Cell-specific TREs from the AFP genehave been identified. For example, the cloning and characterization ofhuman AFP-specific enhancer activity is described in Watanabe et al.(1987) J. Biol. Chem. 262:4812-4818. A 5′ AFP regulatory region(containing the promoter, putative silencer, and enhancer) is containedwithin approximately 5 kb upstream from the transcription start site.

[0167] Within the AFP regulatory region, a human AFP enhancer region islocated between about nt −3954 and about nt −3335, relative to thetranscription start site of the AFP gene. The human AFP promoterencompasses a region from about nt −174 to about nt+29. Juxtapositioningof these two genetic elements, yields a fully functional AFP-TRE. Ido etal. (1995) Cancer Res. 55:3105-3109 describe a 259 bp promoter fragment(nt −230 to nt+29) that is specific for expression in HCC cells. The AFPenhancer, located between nt −3954 and nt −3335 relative to thetranscription start site, contains two regions, denoted A and B. Thepromoter region contains typical TATA and CAAT boxes. Preferably, theAFP-TRE contains at least one enhancer region. More preferably, theAFP-TRE contains both enhancer regions.

[0168] Suitable target cells for vectors containing AFP-TREs are anycell type that allow an AFP-TRE to function. Preferred are cells thatexpress or produce AFP, including, but not limited to, tumor cellsexpressing AFP. Examples of such cells are hepatocellular carcinoma(HCC) cells, gonadal and other germ cell tumors (especially endodermalsinus tumors), brain tumor cells, ovarian tumor cells, acinar cellcarcinoma of the pancreas (Kawamoto et al. (1992) Hepatogastroenterology39:282-286), primary gall bladder tumor (Katsuragi et al. (1989) RinskoHoshasen 34:371-374), uterine endometrial adenocarcinoma cells (Koyamaet al. (1996) Jpn. J. Cancer Res. 87:612-617), and any metastases of theforegoing (which can occur in lung, adrenal gland, bone marrow, and/orspleen). In some cases, metastatic disease to the liver from certainpancreatic and stomach cancers produce AFP. Especially preferred astarget cells for an AFP-TRE are hepatocellular carcinoma cells and anyof their metastases.

[0169] AFP production can be measured (and hence AFP-producing cells canbe identified) using immunoassays standard in the art, such as RIA,ELISA or protein immunoblotting (Western blots) to determine levels ofAFP protein production; and/or RNA blotting (Northern blots) todetermine AFP mRNA levels. Alternatively, such cells can be identifiedand/or characterized by their ability to activate transcriptionally anAFP-TRE (i.e., allow an AFP-TRE to function).

[0170] See also co-owned PCT WO98/39465 regarding AFP-TREs. As describedin more detail therein, an AFP-TRE can comprise any number ofconfigurations, including, but not limited to, an AFP promoter; an AFPenhancer; an AFP promoter and an AFP enhancer; an AFP promoter and aheterologous enhancer; a heterologous promoter and an AFP enhancer; andmultimers of the foregoing. The promoter and enhancer components of anAFP-TRE can be in any orientation and/or distance from the codingsequence of interest, as long as the desired AFP cell-specifictranscriptional activity is obtained. An adenovirus vector of thepresent invention can comprise an AFP-TRE endogenous silencer element orthe AFP-TRE endogenous silencer element can be deleted.

[0171] Urokinase Plasminogen Activator

[0172] The protein urokinase plasminogen activator (uPA) and its cellsurface receptor, urokinase plasminogen activator receptor (uPAR), areexpressed in many of the most frequently-occurring neoplasms and appearto represent important proteins in cancer metastasis. Both proteins areimplicated in breast, colon, prostate, liver, renal, lung and ovariancancer. Sequence elements that regulate uPA and uPAR transcription havebeen extensively studied. Riccio et al. (1985) Nucleic Acids Res.13:2759-2771; Cannio et al. (1991) Nucleic Acids Res. 19:2303-2308.

[0173] Carcinoembryonic Antigen (CEA)

[0174] CEA is a 180,000 Dalton, tumor-associated, glycoprotein antigenpresent on endodermally-derived neoplasms of the gastrointestinal tract,such as colorectal, gastric (stomach) and pancreatic cancer, as well asother adenocarcinomas such as breast and lung cancers. CEA is ofclinical interest because circulating CEA can be detected in the greatmajority of patients with CEA-positive tumors. In lung cancer, about 50%of total cases have circulating CEA, with high concentrations of CEA(greater than 20 ng/ml) often detected in adenocarcinomas. Approximately50% of patients with gastric carcinoma are serologically positive forCEA.

[0175] The 5′-flanking sequence of the CEA gene has been shown to confercell-specific activity. The CEA promoter region, approximately the first424 nucleotides upstream of the transcriptional start site in the 5′flanking region of the gene, was shown to confer cell-specific activityby virtue of providing higher promoter activity in CEA-producing cellsthan in non-producing HeLa cells. Schrewe et al. (1990) Mol. Cell. Biol.10:2738-2748. In addition, cell-specific enhancer regions have beenfound. See PCT/GB/02546 The CEA promoter, putative silencer, andenhancer elements appears to be contained within a region that extendsapproximately 14.5 kb upstream from the transcription start site.Richards et al. (1995); PCT/GB/02546. Further characterization of the5′-flanking region of the CEA gene by Richards et al. (1995) supraindicated that two upstream regions (one between about −13.6 and about−10.7 kb, and the other between about −6.1 and about-4.0 kb), whenlinked to the multimerized promoter, resulted in high-level andselective expression of a reporter construct in CEA-producing LoVo andSW1463 cells. Richards et al. (1995) supra also localized the promoterregion between about nt −90 and about nt+69 relative to thetranscriptional start site, with the region between about nt −41 andabout nt −18 being essential for expression. PCT/GB/02546 describes aseries of 5′-flanking CEA fragments which confer cell-specific activity,including fragments comprising the following sequences: about nt −299 toabout nt+69; about nt −90 to about nt+69; nt −14,500 to nt −10,600; nt−13,600 to nt −10,600; and nt −6100 to nt −3800, with all coordinatesbeing relative to the transcriptional start point. In addition,cell-specific transcription activity is conferred on an operably linkedgene by the CEA fragment from nt −402 to nt+69.

[0176] CEA-TREs for use in the vectors disclosed herein are derived frommammalian cells, including, but not limited to, human cells. Thus, anyof the CEA-TREs can be used as long as the requisite desiredfunctionality is displayed by the vector.

[0177] Mucin

[0178] The protein product of the MUC1 gene (known as mucin, MUC1protein; episialin; polymorphic epithelial mucin or PEM; EMA; DF3antigen; NPGP; PAS-O; or CA15.3 antigen) is normally expressed mainly atthe apical surface of epithelial cells lining the glands or ducts of thestomach, pancreas, lungs, trachea, kidney, uterus, salivary glands, andmammary glands. Zotter et al. (1988) Cancer Rev. 11-12:55-101; andGirling et al. (1989) Int. J. Cancer 43:1072-1076. However, mucin isoverexpressed in 75-90% of human breast carcinomas. Kufe et al. (1984)Hybridoma 3:223-232. For reviews, see Hilkens (1988) Cancer Rev.11-12:25-54; and Taylor-Papadimitriou, et al. (1990) J. Nucl. Med.Allied Sci. 34:144-150. Mucin protein expression correlates with thedegree of breast tumor differentiation. Lundy et al. (1985) BreastCancer Res. Treat. 5:269-276.

[0179] Overexpression of the MUC1 gene in human breast carcinoma cellsMCF-7 and ZR-75-1 appears to occur at the transcriptional level. Kufe etal. (1984) supra; Kovarik (1993) J. Biol. Chem. 268:9917-9926; and Abeet al. (1990) J. Cell. Physiol. 143:226-231. The regulatory sequences ofthe MUC1 gene have been cloned, including the approximately 0.9 kbupstream of the transcription start site which contains a TRE thatappears to be involved in cell-specific transcription. Abe et al. (1993)Proc. Natl. Acad. Sci. USA 90:282-286; Kovarik et al. (1993) supra; andKovarik et al. (1996) J. Biol. Chem. 271:18140-18147.

[0180] MUC1-TREs are derived from mammalian cells, including but notlimited to, human cells. Preferably, the MUC1-TRE is human. In oneembodiment, the MUC1-TRE contains the entire 0.9 kb 5′ flanking sequenceof the MUC1 gene. In other embodiments, MUC1-TREs comprise the followingsequences (relative to the transcription start site of the MUC1 gene)operably-linked to a promoter: about nt −725 to about nt+31, about nt−743 to about nt+33, about nt −750 to about nt+33, and about nt −598 toabout nt+485.

[0181] c-erbB2/HER-2/neu

[0182] The c-erbB2/neu gene (HER-2/neu or HER) is a transforming genethat encodes a 185 kD epidermal growth factor receptor-relatedtransmembrane glycoprotein. In humans, the c-erbB2/neu protein isexpressed during fetal development and, in adults, the protein is weaklydetectable (by immunohistochemistry) in the epithelium of many normaltissues. Amplification and/or over-expression of the c-erbB2/neu genehas been associated with many human cancers, including breast, ovarian,uterine, prostate, stomach and lung cancers. The clinical consequencesof overexpression of the c-erbB2/neu protein have been best studied inbreast and ovarian cancer. c-erbB2/neu protein over-expression occurs in20 to 40% of intraductal carcinomas of the breast and 30% of ovariancancers, and is associated with a poor prognosis in subcategories ofboth diseases.

[0183] Human, rat and mouse c-erbB2/neu TREs have been identified andshown to confer transcriptional activity specific toc-erbB2/neu-expressing cells. Tal et al. (1987) Mol. Cell. Biol.7:2597-2601; Hudson et al. (1990) J. Biol. Chem. 265:4389-4393;Grooteclaes et al. (1994) Cancer Res. 54:4193-4199; Ishii et al. (1987)Proc. Natl. Acad. Sci. USA 84:4374-4378; and Scott et al. (1994) J.Biol. Chem. 269:19848-19858.

[0184] Melanocyte-Specific TRE

[0185] It has been shown that some genes which encode melanoma proteinsare frequently expressed in melanoma/melanocytes, but silent in themajority of normal tissues. A variety of melanocyte-specific TRE areknown, are responsive to cellular proteins (transcription factors and/orco-factor(s)) associated with melanocytes, and comprise at least aportion of a melanocyte-specific promoter and/or a melanocyte-specificenhancer. Known transcription factors that control expression of one ormore melanocyte-specific genes include the microphthalmia associatedtranscription factor MITF. Yasumoto et al. (1997) J. Biol. Chem.272:503-509. Other transcription factors that control expression of oneor more melanocyte specific genes include MART-1/Melan-A, gp100, TRP-1and TRP-2 Methods are described herein for measuring the activity of amelanocyte-specific TRE and thus for determining whether a given cellallows a melanocyte-specific TRE to function.

[0186] In some embodiments, the melanocyte-specific TREs used in thisinvention are derived from mammalian cells, including but not limitedto, human, rat, and mouse. Any melanocyte-specific TREs may be used inthe adenoviral vectors of the invention. Rodent and human 5′ flankingsequences from genes expressed specifically or preferentially inmelanoma cells have been described in the literature and are thus madeavailable for practice of this invention and need not be described indetail herein. The following are some examples of melanocyte-specificTREs which can be used. A promoter and other control elements in thehuman tyrosinase gene 5′ flanking region have been described andsequences have been deposited as GenBank Accession Nos. X16073 andD10751. Kikuchi et al. (1989) Biochim. Biophys. Acta 1009:283-286; andShibata et al. (1992) J. Biol. Chem. 267:20584-20588. A cis-actingelement has been defined that enhances melanocyte-specific expression ofhuman tyrosinase gene. This element comprises a 20-bp sequence known astyrosinase distal element (TDE), contains a CATGTG motif, and lies atpositions about −1874 to about −1835 relative to the human tyrosinasegene transcription start site. Yasumoto et al. (1994) Mol. Cell. Biol.14:8058-8070. A promoter region comprising sequences from about −209 to+61 of the human tyrosinase gene was found to direct melanocyte-specificexpression. Shibata (1992). Similarly, the mouse tyrosinase 5′ flankingregion has been analyzed and a sequence deposited as GenBank AccessionNos. D00439 and X51743. Klüppel et al. (1991) Proc. Natl. Acad. Sci. USA88:3777-3788. A minimal promoter has been identified for the mouse TRP-1gene, and was reported to encompass nucleotides −44 to +107 relative tothe transcription start site. Lowings et al. (1992) Mol. Cell. Biol.12:3653-3662. Two regulatory regions required for melanocyte-specificexpression of the human TRP-2 gene have been identified. Yokoyama et al.(1994) J. Biol. Chem. 269:27080-27087. A human MART-I promoter regionhas been described and deposited as GenBank Accession No. U55231.Melanocyte-specific promoter activity was found in a 233-bp fragment ofthe human MART-1 gene 5′ flanking region. Butterfield et al. (1997) Gene191:129-134. A basic-helix-loop-helix/leucine zipper-containingtranscription factor, MITF (microphthalmia associated transcriptionfactor) was reported to be involved in transcriptional activation oftyrosinase and TRP-1 genes. Yasumoto et al. (1997) J. Biol. Chem.272:503-509.

[0187] In some embodiments, a melanocyte-specific TRE comprisessequences derived from the 5′ flanking region of a human tyrosinase genedepicted in Table 3. In some of these embodiments, themelanocyte-specific TRE comprises tyrosinase nucleotides from about −231to about +65 relative to the transcription start site (from aboutnucleotide 244 to about nucleotide 546 of SEQ ID NO:10) and may furthercomprise nucleotides from about −1956 to about −1716 relative to thehuman tyrosinase transcription start site (from about nucleotide 6 toabout nucleotide 243 of SEQ ID NO:10). A melanocyte-specific TRE cancomprise nucleotides from about—−231 to about +65 juxtaposed tonucleotides from about −1956 to about −1716. It has been reported thatnucleotides from about −1956 to about −1716 relative to the humantyrosinase transcription start site can confer melanocyte-specificexpression of an operably linked reporter gene with either a homologousor a heterologous promoter. Accordingly, in some embodiments, amelanocyte-specific TRE comprises nucleotides from about −1956 to about−1716 operably linked to a heterologous promoter.

[0188] A melanocyte-specific TRE can also comprise multimers. Forexample, a melanocyte-specific TRE can comprise a tandem series of atleast two, at least three, at least four, or at least five tyrosinasepromoter fragments. Alternatively, a melanocyte-specific TRE could haveone or more tyrosinase promoter regions along with one or moretyrosinase enhancer regions. These multimers may also containheterologous promoter and/or enhancer sequences.

[0189] Cell Status-Specific TREs

[0190] Cell status-specific TREs for use in the adenoviral vectors ofthe present invention can be derived from any species, preferably amammal. A number of genes have been described which are expressed inresponse to, or in association with, a cell status. Any of these cellstatus-associated genes may be used to generate a cell status-specificTRE.

[0191] An example of a cell status is cell cycle. An exemplary genewhose expression is associated with cell cycle is E2F-1, a ubiquitouslyexpressed, growth-regulated gene, which exhibits peak transcriptionalactivity in S phase. Johnson et al. (1994) Genes Dev. 8:1514-1525. TheRB protein, as well as other members of the RB family, form specificcomplexes with E2F-1, thereby inhibiting its ability to activatetranscription. Thus, E2F-1-responsive promoters are down-regulated byRB. Many tumor cells have disrupted RB function, which can lead tode-repression of E2F-1-responsive promoters, and, in turn, de-regulatedcell division.

[0192] Accordingly, in one embodiment, the invention provides anE3-containing adenoviral vector in which an adenoviral gene (preferablya gene necessary for replication) is under transcriptional control of acell status-specific TRE, wherein the cell status-specific TRE comprisesa cell cycle-activated TRE. In one embodiment, the cell cycle-activatedTRE is an E2 μl TRE.

[0193] Another group of genes that are regulated by cell status arethose whose expression is increased in response to hypoxic conditions.Bunn and Poyton (1996) Physiol. Rev. 76:839-885; Dachs and Stratford(1996) Br. J. Cancer 74:5126-5132; Guillemin and Krasnow (1997) Cell89:9-12. Many tumors have insufficient blood supply, due in part to thefact that tumor cells typically grow faster than the endothelial cellsthat make up the blood vessels, resulting in areas of hypoxia in thetumor. Folkman (1989) J. Natl. Cancer Inst. 82:4-6; and Kallinowski(1996) The Cancer J. 9:37-40. An important mediator of hypoxic responsesis the transcriptional complex HIF-1, or hypoxia inducible factor-1,which interacts with a hypoxia-responsive element (HRE) in theregulatory regions of several genes, including vascular endothelialgrowth factor, and several genes encoding glycolytic enzymes, includingenolase-1. Murine HRE sequences have been identified and characterized.Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. An HRE froma rat enolase-1 promoter is described in Jiang et al. (1997) Cancer Res.57:5328-5335. An HRE from a rat enolase-1 promoter is depicted in Table3.

[0194] Accordingly, in one embodiment, an adenovirus vector comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a cell status-specific TREcomprising an HRE. In one embodiment, the cell status-specific TREcomprises the HRE depicted in Table 3.

[0195] Other cell status-specific TREs include heat-inducible (i.e.,heat shock) promoters, and promoters responsive to radiation exposure,including ionizing radiation and UV radiation. For example, the promoterregion of the early growth response-1 (Egr-1) gene contains anelement(s) inducible by ionizing radiation. Hallahan et al. (1995) Nat.Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids. Res.16:8835-8846. Heat-inducible promoters, including heat-inducibleelements, have been described. See, for example Welsh (1990) in “StressProteins in Biology and Medicine”, Morimoto, Tisseres, and Georgopoulos,eds. Cold Spring Harbor Laboratory Press; and Perisic et al. (1989) Cell59:797-806. Accordingly, in some embodiments, the cell status-specificTRE comprises an element(s) responsive to ionizing radiation. In oneembodiment, this TRE comprises a 5′ flanking sequence of an Egr-1 gene.In other embodiments, the cell status-specific TRE comprises a heatshock responsive element.

[0196] The cell status-specific TREs listed above are provided asnon-limiting examples of TREs that would function in the instantinvention. Additional cell status-specific TREs are known in the art, asare methods to identify and test cell status specificity of suspectedcell status-specific TREs.

[0197] Urothelial Cell-Specific TREs

[0198] Any urothelial cell-specific TRE may be used in the adenoviralvectors of the invention. A number of urothelial cell-specific proteinshave been described, among which are the uroplakins. Uroplakins (UP),including UPIa and UPIb (27 and 28 kDa, respectively), UPII (15 kDa),and UPIII (47 kDa), are members of a group of integral membrane proteinsthat are major proteins of urothelial plaques. These plaques cover alarge portion of the apical surface of mammalian urothelium and may playa role as a permeability barrier and/or as a physical stabilizer of theurothelial apical surface. Wu et al. (1994) J. Biol. Chem.269:13716-13724. UPs are bladder-specific proteins, and are expressed ona significant proportion of urothelial-derived tumors, including about88% of transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol.147:1383-1397; and Wu et al. (1998) Cancer Res. 58:1291-1297. Thecontrol of the expression of the human UPII has been studied, and a3.6-kb region upstream of the mouse UPII gene has been identified whichcan confer urothelial-specific transcription on heterologous genes (Linet al. (1995) Proc. Natl. Acad. Sci. USA 92:679-683).

[0199] Preferred urothelial cell-specific TREs include TREs derived fromthe uroplakins UPIa, UPIb, UPII, and UPIII, as well as urohingin. Auroplakin TRE may be from any species, depending on the intended use ofthe adenovirus, as well as the requisite functionality is exhibited inthe target or host cell. Significantly, adenovirus constructs comprisinga urothelial cell-specific TREs have observed that such constructs arecapable of selectively replicating in urothelial cells as opposed tosmooth muscle cells, which adjoin urothelial cells in the bladder.

[0200] Uroplakin

[0201] Urothelial-specific TREs derived from the hUPII gene aredescribed herein. Accordingly, in some embodiments, an adenovirus vectorof the invention comprises an adenovirus gene, preferably an adenoviralgene essential for replication, under transcriptional control of aurothelial cell-specific TRE which comprises the 2.2 kb sequence fromthe 5′ flanking region of hUPII gene, as shown in Table 3. In otherembodiments, an adenovirus vector of the invention comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a urothelial cell-specificTRE which comprises a 1.8 kb sequence from the 5′ flanking region ofhUPII gene, from nucleotides 430 to 2239 as shown in Table 3. In otherembodiments, the urothelial cell-specific TRE comprises a functionalportion of the 2.2 kb sequence depicted in Table 3, or a functionalportion of the 1.8 kb sequence of nucleotides 430 to 2239 of thesequence depicted in Table 3, such as a fragment of 2000 bp or less,1500 bp or less, or 1000 bp or less, 600 bp less, or at least 200 bpwhich includes the 200 bp fragment of the hUPII 5′-flanking region.

[0202] A 3.6 kb 5′-flanking sequence located from the mouse UPII (mUPI)gene which confers urothelial cell-specific transcription onheterologous genes is one urothelial cell-specific TRE useful in vectorsof the instant invention (Table 3). Smaller TREs (i.e., 3500 bp or less,more preferably less than about 2000 bp, 1500 bp, or 1000 bp) arepreferred. Smaller TREs derived from the mUPII 3.6 kb fragment are onegroup of preferred urothelial cell-specific TREs. In particular,Inventors have identified an approximately 600 bp fragment from the 5′flanking DNA of the mUPII gene, which contains 540 bp of 5′ untranslatedregion (UTR) of the mUPII gene, that confers urothelial cell-specificexpression on heterologous genes.

[0203] Accordingly, in some embodiments, an adenovirus vector of theinvention comprises an adenovirus gene, preferably an adenoviral geneessential for replication, under transcriptional control of a urothelialcell-specific TRE which comprises the 3.6 kb sequence from the 5′flanking region of mouse UPII gene, as shown in Table 3. In otherembodiments, the urothelial cell-specific TRE comprises a functionalportion of the 3.6 kb sequence depicted in Table 3, such as a fragmentof 3500 bp or less, 2000 bp or less, 1500 bp or less, or 1000 bp or lesswhich includes the 540 bp fragment of 5′ UTR. The urothelialcell-specific TRE may also be a sequence which is substantiallyidentical to the 3.6 kb mUPII 5′-flanking region or any of the describedfragments thereof.

[0204] As an example of how urothelial cell-specific TRE activity can bedetermined, a polynucleotide sequence or set of such sequences can begenerated using methods known in the art, such as chemical synthesis,site-directed mutagenesis, PCR, and/or recombinant methods. Thesequence(s) to be tested is inserted into a vector containing anappropriate reporter gene, including, but not limited to,chloramphenicol acetyl transferase (CAT), β-galactosidase (encoded bythe lacZ gene), luciferase (encoded by the luc gene), a greenfluorescent protein, alkaline phosphatase, and horse radish peroxidase.Such vectors and assays are readily available, from, inter alia,commercial sources. Plasmids thus constructed are transfected into asuitable host cell to test for expression of the reporter gene ascontrolled by the putative target cell-specific TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes (lipofection) and DEAE dextran. Suitable hostcells include any urothelial cell type, including but not limited to,KU-1, MYP3 (a non-tumorigenic rat urothelial cell line), 804G (ratbladder carcinoma cell line), cultured human urothelial cells (HUC),HCV-29, UM-UC-3, SW780, RT4, HL60, KG-1, and KG-1A. Non-urothelialcells, such as LNCaP, HBL-100, HLF, HLE, 3T3, Hep3B, HuH7, CADO-LC9, andHeLa are used as a control. Results are obtained by measuring the levelof expression of the reporter gene using standard assays. Comparison ofexpression between urothelial cells and control indicates presence orabsence of transcriptional activation.

[0205] Comparisons between or among various urothelial cell-specificTREs can be assessed by measuring and comparing levels of expressionwithin a single urothelial cell line. It is understood that absolutetranscriptional activity of a urothelial cell-specific TRE will dependon several factors, such as the nature of the target cell, delivery modeand form of the urothelial cell-specific TRE, and the coding sequencethat is to be selectively transcriptionally activated. To compensate forvarious plasmid sizes used, activities can be expressed as relativeactivity per mole of transfected plasmid. Alternatively, the level oftranscription (i.e., mRNA) can be measured using standard Northernanalysis and hybridization techniques. Levels of transfection (i.e.,transfection efficiencies) are measured by co-transfecting a plasmidencoding a different reporter gene under control of a different TRE,such as the CMV immediate early promoter. This analysis can alsoindicate negative regulatory regions, i.e., silencers.

[0206] Alternatively a putative urothelial cell-specific TRE can beassessed for its ability to confer adenoviral replication preference forcells that allow a urothelial cell-specific TRE to function. For thisassay, constructs containing an adenovirus gene essential to replicationoperatively linked to a putative urothelial cell-specific TRE aretransfected into urothelial cells. Viral replication in those cells iscompared, for example, to viral replication by wild type adenovirus inthose cells and/or viral replication by the construct in non-urothelialcells.

[0207] TRE Configurations

[0208] A TRE as used in the present invention can be present in avariety of configurations. A TRE can comprise multimers. For example, aTRE can comprise a tandem series of at least two, at least three, atleast four, or at least five target cell-specific TREs. These multimersmay also contain heterologous promoter and/or enhancer sequences.

[0209] Optionally, a transcriptional terminator or transcriptional“silencer” can be placed upstream of the target cell-specific TRE, thuspreventing unwanted read-through transcription of the coding segmentunder transcriptional control of the target cell-specific TRE. Also,optionally, the endogenous promoter of the coding segment to be placedunder transcriptional control of the target cell-specific TRE can bedeleted.

[0210] A target cell-specific TRE may or may not lack a silencer. Thepresence of a silencer (i.e., a negative regulatory element) may assistin shutting off transcription (and thus replication) in non-permissivecells (i.e., a non-target cell). Thus, presence of a silencer may conferenhanced target cell-specific replication by more effectively preventingadenoviral vector replication in non-target cells. Alternatively, lackof a silencer may assist in effecting replication in target cells, thusconferring enhanced target cell-specific replication due to moreeffective replication in target cells.

[0211] It is also understood that the invention includes a targetcell-specific TRE regulating the transcription of a bicistronic mRNA inwhich translation of the second mRNA is associated by an IRES. Anadenovirus vector may further include an additional heterologous TREwhich may or may not be operably linked to the same gene(s) as thetarget cell-specific TRE. For example a TRE (such as a celltype-specific or cell status-specific TRE) may be juxtaposed to a secondtype of target-cell-specific TRE. “Juxtaposed” means a targetcell-specific TRE and a second TRE transcriptionally control the samegene. For these embodiments, the target cell-specific TRE and the secondTRE may be in any of a number of configurations, including, but notlimited to, (a) next to each other (i.e., abutting); (b) both 5′ to thegene that is transcriptionally controlled (i.e., may have interveningsequences between them); (c) one TRE 5′ and the other TRE 3′ to thegene.

[0212] As is readily appreciated by one skilled in the art, a targetcell-specific TRE is a polynucleotide sequence, and, as such, canexhibit function over a variety of sequence permutations. Methods ofnucleotide substitution, addition, and deletion are known in the art,and readily available functional assays (such as the CAT or luciferasereporter gene assay) allow one of ordinary skill to determine whether asequence variant exhibits requisite target cell-specific transcriptionfunction. Hence, the invention also includes functionally-preservedvariants of the TRE nucleic acid sequences disclosed herein, whichinclude nucleic acid substitutions, additions, and/or deletions. Thevariants of the sequences disclosed herein may be 80%, 85%, 90%, 95%,98%, 99% or more identical, as measured by, for example, ALIGN Plus(Scientific and Educational Software, Pennsylvania), preferably usingefault parameters, which are as follows: mismatch=2; open gap=0; extendgap=2 to any of the urothelial cell-specific TRE sequences disclosedherein. Variants of target cell-specific TRE sequences may alsohybridize at high stringency, that is at 68° C. and 0.1×SSC, to any ofthe target cell-specific TRE sequences disclosed herein.

[0213] In terms of hybridization conditions, the higher the sequenceidentity required, the more stringent are the hybridization conditionsif such sequences are determined by their ability to hybridize to asequence of TRE disclosed herein. Accordingly, the invention alsoincludes polynucleotides that are able to hybridize to a sequencecomprising at least about 15 contiguous nucleotides (or more, such asabout 25, 35, 50, 75 or 100 contiguous nucleotides) of a TRE disclosedherein. The hybridization conditions would be stringent, i.e., 80° C.(or higher temperature) and 6M SSC (or less concentrated SSC). Anotherset of stringent hybridization conditions is 68° C. and 0.1×SSC. Fordiscussion regarding hybridization reactions, see below.

[0214] Hybridization reactions can be performed under conditions ofdifferent “stringency”. Conditions that increase stringency of ahybridization reaction of widely known and published in the art. See,for example, Sambrook et al. (1989) at page 7.52. Examples of relevantconditions include (in order of increasing stringency): incubationtemperatures of 25° C., 37° C., 50° C. and 68° C.; buffer concentrationsof 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mMcitrate buffer) and their equivalents using other buffer systems;formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from5 minutes to 24 hours; 1, 2, or more washing steps; wash incubationtimes of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC,0.1×SSC, or deionized water. An exemplary set of stringent hybridizationconditions is 68° C. and 0.1×SSC.

[0215] “T_(m)” is the temperature in degrees Celcius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:

T _(m)=81.5+16.6 log[X ⁺]+0.41(% G/C)−0.61 (% F)−600/L

[0216] where [X⁺] is the cation concentration (usually sodium ion, Na⁺)in mol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

[0217] While not wishing to be bound by a single theory, the inventorsnote that it is possible that certain modifications will result inmodulated resultant expression levels, including enhanced expressionlevels. Achievement of modulated resultant expression levels, preferablyenhanced expression levels, may be especially desirable in the case ofcertain, more aggressive forms of cancer, or when a more rapid and/oraggressive pattern of cell killing is warranted (due to animmunocompromised condition of the individual, for example).

[0218] Determination of TRE Activity

[0219] Activity of a TRE can be determined, for example, as follows. ATRE polynucleotide sequence or set of such sequences can be generatedusing methods known in the art, such as chemical synthesis,site-directed mutagenesis, PCR, and/or recombinant methods. Thesequence(s) to be tested can be inserted into a vector containing apromoter (if no promoter element is present in the TRE) and anappropriate reporter gene encoding a reporter protein, including, butnot limited to, chloramphenicol acetyl transferase (CAT),β-galactosidase (encoded by the lacZ gene), luciferase (encoded by theluc gene), alkaline phosphatase (AP), green fluorescent protein (GFP),and horseradish peroxidase (HRP). Such vectors and assays are readilyavailable, from, inter alia, commercial sources. Plasmids thusconstructed are transfected into a suitable host cell to test forexpression of the reporter gene as controlled by the putative TRE usingtransfection methods known in the art, such as calcium phosphateprecipitation, electroporation, liposomes, DEAE dextran-mediatedtransfer, particle bombardment or direct injection. TRE activity ismeasured by detection and/or quantitation of reporter gene-derived mRNAand/or protein. Reporter protein product can be detected directly (e.g.,immunochemically) or through its enzymatic activity, if any, using anappropriate substrate. Generally, to determine cell specific activity ofa TRE, a TRE-reporter gene construct is introduced into a variety ofcell types. The amount of TRE activity is determined in each cell typeand compared to that of a reporter gene construct lacking the TRE. A TREis determined to be cell-specific if it is preferentially functional inone cell type, compared to a different type of cell.

[0220] Adenovirus Early Genes

[0221] The adenovirus vectors of the invention comprise two or moregenes which are co-transcribed under the control of a targetcell-specific TRE wherein the second gene is under translational controlof an IRES. One or more of the genes can be an adenovirus gene,preferably an adenovirus gene essential for replication. Any gene thatis essential for adenovirus replication, such as E1A, E1B, E2, E4 or anyof the late genes, is useful. The adenovirus may also comprise E3. Inaddition, one or more of the genes can be a transgene or heterologousgene. Any of the various adenovirus serotypes can be used, such as, forexample, Ad2, Ad5, Ad12 and Ad40. For purposes of illustration, the Ad5serotype is exemplified herein.

[0222] The E1A gene is expressed immediately (between 0 and 2 hours)after viral infection, before any other viral genes. E1A protein is atrans-acting positive transcriptional regulatory factor, and is requiredfor the expression of the other early viral genes E1B, E2, E3, E4, andthe promoter-proximal major late genes. Despite the nomenclature, thepromoter proximal genes driven by the major late promoter are alsoexpressed during early times after Ad5 infection. Flint (1982) Biochem.Biophys. Acta 651:175-208; Flint (1986) Advances Virus Research31:169-228; and Grand (1987) Biochem. J. 241:25-38. In the absence of afunctional E1A gene, viral infection does not proceed, because the geneproducts necessary for viral DNA replication are not produced. Nevins(1989) Adv. Virus Res. 31:35-81. The transcription start site of Ad5 E1Ais at coordinate 498 and the ATG start site of the E1A protein is atcoordinate 560 in the virus genome.

[0223] The E1B protein is necessary in trans for transport of late mRNAfrom the nucleus to the cytoplasm. Defects in E1B expression result inpoor expression of late viral proteins and an inability to shut off hostcell protein synthesis. The promoter of E1B has been implicated as thedefining element of difference in the host range of Ad40 and Ad5:clinically Ad40 is an enterovirus, whereas Ad5 causes acuteconjunctivitis. Bailey et al. (1993) Virology 193:631; Bailey et al.(1994) Virology 202:695-706. The E1B promoter of Ad5 consists of asingle high-affinity recognition site for Sp1 and a TATA box, andextends from Ad5 nt 1636 to 1701.

[0224] Adenovirus E1B 19-kDa (19K) protein is a potent inhibitor ofapoptosis and cooperates with E1A to produce oncogenic transformation ofprimary cells (Rao, et al., 1992, Cell Biology, 89:7742-7746). Duringproductive adenovirus infection, E1A stimulates host cell DNA synthesis,thereby causing cells to aberrantly go through the cell cycle. Inresponse to cell cycle deregulation, the host cell undergoes apoptosis.As a defense mechanism, the E1B 19-kDa protein inhibits this E1A-inducedapoptosis and allows assembly of viral progeny to be completed beforethe cell commits suicide. E1B 19-kDa conducts anti-apoptotic function bymultiple mechanisms. E1B 19-kDa inhibits the apoptosis of multiplestimuli, including E1a, p53 and TNF, for example. According to wild-typeAd5, the E1B 19-kDa region is located between nucleotide 1714 andnucleotide 2244. The E1B 19-kDa region has been described in, forexample, Rao et al., Proc. Natl. Acad. Sci. USA, 89:7742-7746.

[0225] In a preferred embodiment, expression of the E1A and E1B regionsof the Ad genome is facilitated in a cell-specific fashion by placing acell-specific TRE upstream of E1A and a internal ribosome entry sitebetween E1A and E1B.

[0226] The E2 region of adenovirus encodes proteins related toreplication of the adenoviral genome, including the 72 kD DNA-bindingprotein, the 80 kD precursor terminal protein and the viral DNApolymerase. The E2 region of Ad5 is transcribed in a rightwardorientation from two promoters, termed E2 early and E2 late, mapping at76.0 and 72.0 map units, respectively. While the E2 late promoter istransiently active during late stages of infection and is independent ofthe E1A transactivator protein, the E2 early promoter is crucial duringthe early phases of viral replication.

[0227] The E2 early promoter of Ad5 is located between nucleotides27,050 and 27,150, and consists of a major and a minor transcriptioninitiation site (the latter accounting for about 5% of E2 transcripts),two non-canonical TATA boxes, two E2F transcription factor binding sitesand an ATF transcription factor binding site. For a detailed review ofE2 promoter architecture see Swaminathan et al. (1995) Curr. Topics inMicro. and Imm. 199 part 3:177-194.

[0228] The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable for geneticmanipulation. However, the E2 early promoter overlaps by only a few basepairs with sequences on the counterstrand which encode a 33 kD protein.Notably, an SpeI restriction site (Ad5 position 27,082) is part of thestop codon for the above mentioned 33 kD protein and convenientlyseparates the major E2 early transcription initiation site and TATA boxfrom the upstream E2F and ATF binding sites. Therefore, insertion of aheterologous TRE having SpeI ends into the SpeI site disrupts theendogenous E2 early promoter of Ad5 and allows TRE-regulated expressionof E2 transcripts.

[0229] An E3 region refers to the region of the adenoviral genome thatencodes the E3 products. The E3 region has been described in variouspublications, including, for example, Wold et al. (1995) Curr. TopicsMicrobiol. Immunol. 199:237-274. Generally, the E3 region is locatedbetween about 28583 and about 30470 of the adenoviral genome. An E3region for use in the present invention may be from any adenovirusserotype. An E3 sequence is a polynucleotide sequence that contains asequence from an E3 region. In some embodiments, the sequence encodesADP. In other embodiments, the sequence encodes other than ADP andexcludes a sequence encoding only ADP. As is well known in the art, theADP coding region is located in the E3 region within the adenoviralgenome from about 29468 bp to about 29773 bp; including the Y leader,the location of ADP is from about 28375 bp to about 29773 bp for Ad5.Other ADP regions for other serotypes are known in the art. An E3sequence includes, but is not limited to, deletions; insertions;fusions; and substitutions. An E3 sequence may also comprise an E3region or a portion of the E3 region. It is understood that, as an “E3sequence” is not limited to an “E3 region”, alternative referencesherein to an “E3 region” or “E3 sequence” do not indicate that theseterms are interchangeable. Assays for determining a functional E3sequence for purposes of this invention are described herein.

[0230] The E4 gene has a number of transcription products and encodestwo polypeptides (the products of open reading frames (ORFs) 3 and 6)which are responsible for stimulating the replication of viral genomicDNA and stimulating late gene expression, through interaction withheterodimers of cellular transcription factors E2F-1 and DP-1. The ORF 6protein requires interaction with the E1B 55 kD protein for activitywhile the ORF 3 protein does not. In the absence of functional ORF 3-and ORF 6-encoded proteins, efficiency of plaque formation is less than10⁻⁶ that of wild type virus.

[0231] To further increase cell-specificity of replication, it ispossible to take advantage of the interaction between the E4 ORF 6 geneproduct and the E1B 55 kD protein. For example, if E4 ORFs 1-3 aredeleted, viral DNA replication and late gene synthesis becomes dependenton E4 ORF6 protein. By generating such a deletion in a vector in whichthe E1B region is regulated by a cell-specific TRE, a virus is obtainedin which both E1B and E4 functions are dependent on the cell-specificTRE which regulates E1B.

[0232] Late genes relevant to the disclosed vectors are L1, L2 and L3,which encode proteins of the virion. All of these genes (typicallycoding for structural proteins) are probably required for adenoviralreplication. All late genes are under the control of the major latepromoter (MLP), which is located in Ad5 between nucleotides 5986 and6048.

[0233] In one embodiment, an adenovirus early gene is undertranscriptional control of a cell specific, heterologous TRE. Inadditional embodiments, the early gene is selected from the groupincluding E1A, E1B, E2, E3, E4. In another embodiment, an adenoviruslate gene is under transcriptional control of a cell specific,heterologous TRE. In further embodiments, two or more early genes areunder the control of heterologous TREs that function in the same targetcell. The heterologous TREs can be the same or different, or one can bea variant of the other. In additional embodiments, two or more lategenes are under the control of heterologous TREs that function in thesame target cell. The heterologous TREs can be the same or different, orone can be a variant of the other. In yet another embodiment, one ormore early gene(s) and one or more late gene(s) are undertranscriptional control of the same or different heterologous TREs,wherein the TREs function in the same target cell.

[0234] In some embodiments of the present invention, the adenovirusvector comprises the essential gene E1A and the E1A promoter is deleted.In other embodiments, the adenovirus vector comprises the essential geneE1A and the E1A enhancer I is deleted. In yet other embodiments, the E1Apromoter is deleted and E1A enhancer I is deleted. In other embodiments,an internal ribosome entry site (IRES) is inserted upstream of E1B (sothat E1B is translationally linked), and a target cell-specific TRE isoperably linked to E1A. In still other embodiments, an (IRES) isinserted upstream of E1B (so that E1B is translationally linked), andtarget cell-specific TRE is operably linked to E1A, which may or may notmaintain the E1A promoter and/or enhancer I (i.e., the E1A promoterand/or enhancer I may be, but not necessarily be, deleted). In yet otherembodiments, the 19-kDa region of E1B is deleted.

[0235] For adenovirus vectors comprising a second gene under control ofan IRES, it is preferred that the endogenous promoter of a gene undertranslational control of an IRES be deleted so that the endogenouspromoter does not interfere with transcription of the second gene. It ispreferred that the second gene be in frame with the IRES if the IREScontains an initiation codon. If an initiation codon, such as ATG, ispresent in the IRES, it is preferred that the initiation codon of thesecond gene is removed and that the IRES and second gene are in frame.Alternatively, if the IRES does not contain an initiation codon or ifthe initiation codon is removed from the IRES, the initiation codon ofthe second gene is used.

[0236] Adenovirus Death Protein (ADP) Gene and Gene Product

[0237] In the construction of adenovirus vectors, the E3 region is oftendeleted to facilitate insertion of one or more TREs and/or transgenes.In some embodiments, however, the adenovirus death protein (ADP),encoded within the E3 region, is retained in an adenovirus vector. TheADP gene, under control of the major late promoter (MLP), appears tocode for a protein (ADP) that is important in expediting host celllysis. Tollefson et al. (1992) J. Virol. 66:3633; and Tollefson et al.(1996) J. Virol. 70:2296. Thus, inclusion of an ADP gene in a viralvector can render the vector more potent, making possible more effectivetreatment and/or a lower dosage requirement.

[0238] An ADP coding sequence is obtained preferably from Ad2 (sincethis is the strain in which the ADP has been most fully characterized)using techniques known in the art, such as PCR. Preferably, the Y leader(which is an important sequence for correct expression of late genes) isalso obtained and placed in operative linkage to the ADP codingsequence. The ADP coding sequence (with or without the Y leader) is thenintroduced into an adenoviral genome, for example, in the E3 region,where expression of the ADP coding sequence will be driven by the MLP.The ADP coding sequence can, of course, also be inserted in otherlocations of the adenovirus genome, such as the E4 region.Alternatively, the ADP coding sequence can be operably linked to aheterologous TRE, including, but not limited to, another viral TRE or atarget cell-specific TRE (see infra). In another embodiment, the ADPgene is present in a viral genome such that it is transcribed as part ofa multi-cistronic mRNA in which its translation is associated with anIRES.

[0239] E3-Containing Target Cell-Specific Adenoviral Vectors

[0240] In some embodiments, the adenovirus vectors contain an E3 region,or a portion of an E3 region. Inclusion of the E3 region of adenoviruscan enhance cytotoxicity of the target cell-specific adenoviral vectorsof the present invention. Adenoviral vectors containing an E3 region maymaintain their high level of specificity and can be (a) significantlymore cytotoxic; (b) produce higher virus yield including extracellularvirus yield; (c) form larger plaques; (d) produce rapid cell death; and(e) kill tumors more efficiently in vivo than vectors lacking the E3region. The adenoviral vectors of this invention may contain the E3region or a portion of the E3 region. It is understood that, asinclusion of E3 confers observable and measurable functionality on theadenoviral vectors, for example, increased replication and production,functionally equivalent (in which functionality is essentiallymaintained, preserved, or even enhanced or diminished) variants of E3may be constructed. For example, portions of E3 may be used. A portionmay be, non-inclusively, either of the following: (a) deletion,preferably at the 3′ end; (b) inclusion of one or more various openreading frames of E3. Five proteins which are encoded by the Ad-E3region have been identified and characterized: (1) a 19-kDa glycoprotein(gp19k) is one of the most abundant adenovirus early proteins, and isknown to inhibit transport of the major histocompatibility complex classI molecules to the cell surface, thus impairing both peptide recognitionand clearance of Ad-infected cells by cytotoxic T lymphocytes (CTLs);(2) E3 14.7k protein and the E3 10.4k/14.5k complex of proteins inhibitthe cytotoxic and inflammatory responses mediated by tumor necrosisfactor (TNF); (3) E3 10.4k/14.5k protein complex down regulates theepidermal growth factor receptor, which may inhibit inflammation andactivate quiescent infected cells for efficient virus replication; (4)E3 11.6k protein (adenoviral death protein, ADP) from adenovirus 2 and 5appears to promote cell death and release of virus from infected cells.The functions of three E3-encoded proteins—3.6k, 6.7k and 12.5k—areunknown. A ninth protein having a molecular weight of 7.5 kDa has beenpostulated to exist, but has not been detected in cells infected withwild-type adenovirus. Wold et al. (1995) Curr. Topics Microbiol.Immunol. 199:237-274. The E3 region is schematically depicted in FIG. 6.These intact, portions, or variants of E3 may be readily constructedusing standard knowledge and techniques in the art. Preferably, anintact E3 region is used.

[0241] In the adenovirus vectors of the present invention, E3 may or maynot be under transcriptional control of native adenoviraltranscriptional control element(s). The E3 promoter is located withinthe coding sequence for virion protein VIII, an essential protein whichis highly conserved among adenovirus serotypes. In some embodiments, E3is under transcriptional control of a heterologous TRE, including, butnot limited to, a target cell-specific TRE. Accordingly, in oneembodiment, the invention provides an adenoviral vector, preferablyreplication competent, that comprises E3 region (or a portion of E3)under transcriptional control of a target cell-specific TRE. In otherembodiments, the E3 region is under transcriptional control of a nativeadenoviral TRE, and the vector further comprises an adenoviral geneessential for replication under transcriptional control of a targetcell-specific TRE. In other embodiments, the E3 region is undertranscriptional control of a target cell-specific TRE, and the vectorfurther comprises an adenoviral gene essential for replication undertranscriptional control of a target cell-specific TRE.

[0242] Transgenes Under Transcriptional Control of a TargetCell-Specific TRE

[0243] Various other replication-competent adenovirus vectors can bemade according to the present invention in which, in addition to havinga single or multiple adenovirus gene(s) under control of a targetcell-specific TRE, a transgene(s) is/are also under control of a targetcell-specific TRE and optionally under translational control of an IRES.Transgenes include, but are not limited to, therapeutic transgenes andreporter genes.

[0244] Reporter Genes

[0245] For example, a target cell-specific TRE can be introduced into anadenovirus vector immediately upstream of and operably linked to anearly gene such as E1A or E1B, and this construct may further comprise asecond co-transcribed gene under translational control of an IRES. Thesecond gene may be a reporter gene. The reporter gene can encode areporter protein, including, but not limited to, chloramphenicol acetyltransferase (CAT), β-galactosidase (encoded by the lacZ gene),luciferase, alkaline phosphatase, a green fluorescent protein, and horseradish peroxidase. For detection of a putative cancer cell(s) in abiological sample, the biological sample may be treated with modifiedadenoviruses in which a reporter gene (e.g., luciferase) is undercontrol of a target cell-specific TRE. The target cell-specific TRE willbe transcriptionally active in cells that allow the target cell-specificTRE to function, and luciferase will be produced. This production willallow detection of target cells, including cancer cells in, for example,a human host or a biological sample. Alternatively, an adenovirus can beconstructed in which a gene encoding a product conditionally requiredfor survival (e.g., an antibiotic resistance marker) is undertranscriptional control of a target cell-specific TRE. When thisadenovirus is introduced into a biological sample, the target cells willbecome antibiotic resistant. An antibiotic can then be introduced intothe medium to kill the non-cancerous cells.

[0246] Therapeutic Transgenes

[0247] Transgenes also include genes which may confer a therapeuticeffect, such as enhancing cytotoxicity so as to eliminate unwantedtarget cells. In this way, various genetic capabilities may beintroduced into target cells, particularly cancer cells. For example, incertain instances, it may be desirable to enhance the degree and/or rateof cytotoxic activity, due to, for example, the relatively refractorynature or particular aggressiveness of the cancerous target cell. Thiscould be accomplished by coupling the target cell-specific cytotoxicactivity with cell-specific expression of, for example, HSV-tk and/orcytosine deaminase (cd), which renders cells capable of metabolizing5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil(5-FU). Using these types of transgenes may also confer a bystandereffect.

[0248] Other desirable transgenes that may be introduced via anadenovirus vector(s) include genes encoding cytotoxic proteins, such asthe A chains of diphtheria toxin, ricin or abrin (Palmiter et al. (1987)Cell 50: 435; Maxwell et al. (1987) Mol. Cell. Biol. 7: 1576; Behringeret al. (1988) Genes Dev. 2: 453; Messing et al. (1992) Neuron 8: 507;Piatak et al. (1988) J. Biol. Chem. 263: 4937; Lamb et al. (1985) Eur.J. Biochem. 148: 265; Frankel et al. (1989) Mol. Cell. Biol. 9: 415),genes encoding a factor capable of initiating apoptosis, sequencesencoding antisense transcripts or ribozymes, which among othercapabilities may be directed to mRNAs encoding proteins essential forproliferation, such as structural proteins, or transcription factors;viral or other pathogenic proteins, where the pathogen proliferatesintracellularly; genes that encode an engineered cytoplasmic variant ofa nuclease (e.g. RNase A) or protease (e.g. awsin, papain, proteinase K,carboxypeptidase, etc.), or encode the Fas gene, and the like. Othergenes of interest include cytokines, antigens, transmembrane proteins,and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-α,-β, -χ, TNF-α, -β, TGF-α, -β, NGF, and the like. The positive effectorgenes could be used in an earlier phase, followed by cytotoxic activitydue to replication.

[0249] Host Cells

[0250] The present invention also provides host cells comprising (i.e.,transformed with) the adenoviral vectors described herein. Bothprokaryotic and eukaryotic host cells can be used as long as sequencesrequisite for maintenance in that host, such as appropriate replicationorigin(s), are present. For convenience, selectable markers are alsoprovided. Host systems are known in the art and need not be described indetail herein. Prokaryotic host cells include bacterial cells, forexample, E. coli, B. subtilis, and mycobacteria. Among eukaryotic hostcells are yeast, insect, avian, plant, C. elegans (or nematode) andmammalian host cells. Examples of fungi (including yeast) host cells areS. cerevisiae, Kluyveromyces lactis (K. lactis), species of Candidaincluding C. albicans and C. glabrata, Aspergillus nidulans,Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowialipolytica. Examples of mammalian cells are cultured human target cells(HUC), KU-1, MYP3 (a non-tumorigenic rat target cell line), 804G (ratbladder carcinoma cell line), HCV-29, UM-UC-3, SW780, RT4, HL60, KG-1,and KG-1A. COS cells, mouse L cells, LNCaP cells, Chinese hamster ovary(CHO) cells, human embryonic kidney (HEK) cells, and African greenmonkey cells. Xenopus laevis oocytes, or other cells of amphibianorigin, may also be used.

[0251] Compositions and Kits

[0252] The present invention also includes compositions, includingpharmaceutical compositions, containing the adenoviral vectors describedherein. Such compositions are useful for administration in vivo, forexample, when measuring the degree of transduction and/or effectivenessof cell killing in an individual. Compositions can comprise anadenoviral vector(s) of the invention and a suitable solvent, such as aphysiologically acceptable buffer. These are well known in the art. Inother embodiments, these compositions further comprise apharmaceutically acceptable excipient. These compositions, which cancomprise an effective amount of an adenoviral vector of this inventionin a pharmaceutically acceptable excipient, are suitable for systemic orlocal administration to individuals in unit dosage forms, sterileparenteral solutions or suspensions, sterile non-parenteral solutions ororal solutions or suspensions, oil in water or water in oil emulsionsand the like. Formulations for parenteral and nonparenteral drugdelivery are known in the art and are set forth in Remington'sPharmaceutical Sciences, 19th Edition, Mack Publishing (1995).Compositions also include lyophilized and/or reconstituted forms of theadenoviral vectors (including those packaged as a virus, such asadenovirus) of the invention.

[0253] The present invention also encompasses kits containing anadenoviral vector(s) of this invention. These kits can be used fordiagnostic and/or monitoring purposes, preferably monitoring. Proceduresusing these kits can be performed by clinical laboratories, experimentallaboratories, medical practitioners, or private individuals. Kitsembodied by this invention allow someone to detect the presence ofbladder cancer cells in a suitable biological sample, such as biopsyspecimens.

[0254] The kits of the invention comprise an adenoviral vector describedherein in suitable packaging. The kit may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, instructions, and interpretive information.

[0255] Preparation of the Adenovirus Vectors of the Invention

[0256] The adenovirus vectors of this invention can be prepared usingrecombinant techniques that are standard in the art. Generally, a targetcell-specific TRE is inserted 5′ to the adenoviral gene of interest,preferably an adenoviral replication gene, more preferably one or moreearly replication genes (although late gene(s) can be used). A targetcell-specific TRE can be prepared using oligonucleotide synthesis (ifthe sequence is known) or recombinant methods (such as PCR and/orrestriction enzymes). Convenient restriction sites, either in thenatural adeno-DNA sequence or introduced by methods such as PCR orsite-directed mutagenesis, provide an insertion site for a targetcell-specific TRE. Accordingly, convenient restriction sites forannealing (i.e., inserting) a target cell-specific TRE can be engineeredonto the 5′ and 3′ ends of a UP-TRE using standard recombinant methods,such as PCR.

[0257] Polynucleotides used for making adenoviral vectors of thisinvention may be obtained using standard methods in the art, such aschemical synthesis, recombinant methods and/or obtained from biologicalsources.

[0258] Adenoviral vectors containing all replication-essential elements,with the desired elements (e.g., E1A) under control of a targetcell-specific TRE, are conveniently prepared by homologous recombinationor in vitro ligation of two plasmids, one providing the left-handportion of adenovirus and the other plasmid providing the right-handregion, one or more of which contains at least one adenovirus gene undercontrol of a target cell-specific TRE. If homologous recombination isused, the two plasmids should share at least about 500 bp of sequenceoverlap. Each plasmid, as desired, may be independently manipulated,followed by cotransfection in a competent host, providing complementinggenes as appropriate, or the appropriate transcription factors forinitiation of transcription from a target cell-specific TRE forpropagation of the adenovirus. Plasmids are generally introduced into asuitable host cell such as 293 cells using appropriate means oftransduction, such as cationic liposomes. Alternatively, in vitroligation of the right and left-hand portions of the adenovirus genomecan also be used to construct recombinant adenovirus derivativecontaining all the replication-essential portions of adenovirus genome.Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020; Bridge et al.(1989) J. Virol. 63: 631-638.

[0259] For convenience, plasmids are available that provide thenecessary portions of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene19:33-42) contains the wild-type left-hand end of Ad5. pBHG10 (Bett etal. (1994); Microbix Biosystems Inc., Toronto) provides the right-handend of Ad5, with a deletion in E3. The deletion in E3 provides room inthe virus to insert a 3 kb target cell-specific TRE without deleting theendogenous enhancer/promoter. The gene for E3 is located on the oppositestrand from E4 (r-strand). pBHG11 provides an even larger E3 deletion(an additional 0.3 kb is deleted). Bett et al. (1994). Alternatively,the use of pBHGE3 (Microbix Biosystems, Inc.) provides the right handend of Ad5, with a full-length of E3.

[0260] For manipulation of the early genes, the transcription start siteof Ad5 E1A is at 498 and the ATG start site of the E1A coding segment isat 560 in the virus genome. This region can be used for insertion of atarget cell-specific TRE. A restriction site may be introduced byemploying polymerase chain reaction (PCR), where the primer that isemployed may be limited to the Ad5 genome, or may involve a portion ofthe plasmid carrying the Ad5 genomic DNA. For example, where pBR322 isused, the primers may use the EcoRI site in the pBR322 backbone and theXbaI site at nt 1339 of Ad5. By carrying out the PCR in two steps, whereoverlapping primers at the center of the region introduce a nucleotidesequence change resulting in a unique restriction site, one can providefor insertion of target cell-specific TRE at that site.

[0261] A similar strategy may also be used for insertion of a targetcell-specific TRE element to regulate E1B. The E1B promoter of Ad5consists of a single high-affinity recognition site for Sp1 and a TATAbox. This region extends from Ad5 nt 1636 to 1701. By insertion of atarget cell-specific TRE in this region, one can provide forcell-specific transcription of the E1B gene. By employing the left-handregion modified with the cell-specific response element regulating E1A,as the template for introducing a target cell-specific TRE to regulateE1B, the resulting adenovirus vector will be dependent upon thecell-specific transcription factors for expression of both E1A and E1B.In additional embodiments, the 19-kDa region of E1B is deleted.

[0262] Similarly, a target cell-specific TRE can be inserted upstream ofthe E2 gene to make its expression cell-specific. The E2 early promoter,mapping in Ad5 from 27050-27150, consists of a major and a minortranscription initiation site, the latter accounting for about 5% of theE2 transcripts, two non-canonical TATA boxes, two E2F transcriptionfactor binding sites and an ATF transcription factor binding site (for adetailed review of the E2 promoter architecture see Swaminathan et al.,Curr. Topics in Micro. and Immunol. (1995) 199(part 3):177-194.

[0263] The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable for geneticmanipulation. However, the E2 early promoter overlaps only for a fewbase pairs with sequences coding for a 33 kD protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kD protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbinding sites E2F and ATF. Therefore, insertion of a targetcell-specific TRE having SpeI ends into the SpeI site in the 1-strandwould disrupt the endogenous E2 early promoter of Ad5 and should allowtarget cell-restricted expression of E2 transcripts.

[0264] For E4, one must use the right hand portion of the adenovirusgenome. The E4 transcription start site is predominantly at about nt35605, the TATA box at about nt 35631 and the first AUG/CUG of ORF I isat about nt 35532. Virtanen et al. (1984) J. Virol. 51: 822-831. Usingany of the above strategies for the other genes, a UP-TRE may beintroduced upstream from the transcription start site. For theconstruction of full-length adenovirus with a target cell-specific TREinserted in the E4 region, the co-transfection and homologousrecombination are performed in W162 cells (Weinberg et al. (1983) Proc.Natl. Acad. Sci. 80:5383-5386) which provide E4 proteins in trans tocomplement defects in synthesis of these proteins.

[0265] Adenoviral constructs containing an E3 region can be generatedwherein homologous recombination between an E3-containing adenoviralplasmid, for example, BHGE3 (Microbix Biosystems Inc., Toronto) and anon-E3-containing adenoviral plasmid, is carried out.

[0266] Alternatively, an adenoviral vector comprising an E3 region canbe introduced into cells, for example 293 cells, along with anadenoviral construct or an adenoviral plasmid construct, where they canundergo homologous recombination to yield adenovirus containing an E3region. In this case, the E3-containing adenoviral vector and theadenoviral construct or plasmid construct contain complementary regionsof adenovirus, for example, one contains the left-hand and the othercontains the right-hand region, with sufficient sequence overlap as toallow homologous recombination.

[0267] Alternatively, an E3-containing adenoviral vector of theinvention can be constructed using other conventional methods includingstandard recombinant methods (e.g., using restriction nucleases and/orPCR), chemical synthesis, or a combination of any of these. Further,deletions of portions of the E3 region can be created using standardtechniques of molecular biology.

[0268] Insertion of an IRES into a vector is accomplished by methods andtechniques that are known in the art and described herein supra,including but not limited to, restriction enzyme digestion, ligation,and PCR. A DNA copy of an IRES can be obtained by chemical synthesis, orby making a cDNA copy of, for example, a picornavirus IRES. See, forexample, Duke et al. (1995) J. Vvirol. 66(3):1602-9) for a descriptionof the EMCV IRES and Huez et al. (1998), Mol. Cell. Biol.18(11):6178-90) for a description of the VEGF IRES. The internaltranslation initiation sequence is inserted into a vector genome at asite such that it lies upstream of a 5′-distal coding region in amulticistronic mRNA. For example, in a preferred embodiment of anadenovirus vector in which production of a bicistronic E1A-E1B mRNA isunder the control of a target cell-specific TRE, the E1B promoter isdeleted or inactivated, and an IRES sequence is placed between E1A andE1B. IRES sequences of cardioviruses and certain aphthoviruses containan AUG codon at the 3′ end of the IRES that serves as both a ribosomeentry site and as a translation initiation site. Accordingly, this typeof IRES is introduced into a vector so as to replace the translationinitiation codon of the protein whose translation it regulates. However,in an IRES of the entero/rhinovirus class, the AUG at the 3′ end of theIRES is used for ribosome entry only, and translation is initiated atthe next downstream AUG codon. Accordingly, if an entero/rhinovirus IRESis used in a vector for translational regulation of a downstream codingregion, the AUG (or other translation initiation codon) of thedownstream gene is retained in the vector construct.

[0269] Methods of packaging polynucleotides into adenovirus particlesare known in the art and are also described in co-owned PCTPCT/US98/04080.

[0270] Delivery of Adenovirus Vectors

[0271] The adenoviral vectors can be used in a variety of forms,including, but not limited to, naked polynucleotide (usually DNA)constructs. Adenoviral vectors can, alternatively, comprisepolynucleotide constructs that are complexed with agents to facilitateentry into cells, such as cationic liposomes or other cationic compoundssuch as polylysine; packaged into infectious adenovirus particles (whichmay render the adenoviral vector(s) more immunogenic); packaged intoother particulate viral forms such as HSV or AAV; complexed with agents(such as PEG) to enhance or dampen an immune response; complexed withagents that facilitate in vivo transfection, such as DOTMA™, DOTAP™, andpolyamines.

[0272] If an adenoviral vector comprising an adenovirus polynucleotideis packaged into a whole adenovirus (including the capsid), theadenovirus itself may also be selected to further enhance targeting. Forexample, adenovirus fibers mediate primary contact with cellularreceptor(s) aiding in tropism. See, e.g., Amberg et al. (1997) Virol.227:239-244. If a particular subgenus of an adenovirus serotypedisplayed tropism for a target cell type and/or reduced affinity fornon-target cell types, such subgenus (or subgenera) could be used tofurther increase cell-specificity of cytotoxicity and/or cytolysis.

[0273] The adenoviral vectors may be delivered to the target cell in avariety of ways, including, but not limited to, liposomes, generaltransfection methods that are well known in the art, such as calciumphosphate precipitation, electroporation, direct injection, andintravenous infusion. The means of delivery will depend in large part onthe particular adenoviral vector (including its form) as well as thetype and location of the target cells (i.e., whether the cells are invitro or in vivo).

[0274] If used in packaged adenoviruses, adenovirus vectors may beadministered in an appropriate physiologically acceptable carrier at adose of about 10⁴ to about 10¹⁴. The multiplicity of infection willgenerally be in the range of about 0.001 to 100. If administered as apolynucleotide construct (i.e., not packaged as a virus) about 0.01 μgto about 1000 μg of an adenoviral vector can be administered. Theadenoviral vector(s) may be administered one or more times, dependingupon the intended use and the immune response potential of the host ormay be administered as multiple, simultaneous injections. If an immuneresponse is undesirable, the immune response may be diminished byemploying a variety of immunosuppressants, so as to permit repetitiveadministration, without a strong immune response. If packaged as anotherviral form, such as HSV, an amount to be administered is based onstandard knowledge about that particular virus (which is readilyobtainable from, for example, published literature) and can bedetermined empirically.

[0275] Methods Using the Adenovirus Vectors of the Invention

[0276] The subject vectors can be used for a wide variety of purposes,which will vary with the desired or intended result. Accordingly, thepresent invention includes methods using the adenoviral vectorsdescribed above.

[0277] In one embodiment, methods are provided for conferring selectivecytotoxicity in cells that allow a target cell-specific TRE to function,preferably target cells, comprising contacting such cells with anadenovirus vector described herein. Cytotoxicity can be measured usingstandard assays in the art, such as dye exclusion, ³H-thymidineincorporation, and/or lysis.

[0278] In another embodiment, methods are provided for propagating anadenovirus specific for cells which allow a target cell-specific TRE tofunction, preferably target cells, preferably cancer cells. Thesemethods entail combining an adenovirus vector with the cells, wherebysaid adenovirus is propagated.

[0279] Another embodiment provides methods for killing cells that allowa target cell-specific TRE to function in a mixture of cells, comprisingcombining the mixture of cells with an adenovirus vector of the presentinvention. The mixture of cells is generally a mixture of normal cellsand cancerous cells that allow a target cell-specific TRE to function,and can be an in vivo mixture or in vitro mixture.

[0280] The invention also includes methods for detecting cells whichallow a target cell-specific TRE to function, such as cancer cells, in abiological sample. These methods are particularly useful for monitoringthe clinical and/or physiological condition of an individual (i.e.,mammal), whether in an experimental or clinical setting. In one method,cells of a biological sample are contacted with an adenovirus vector,and replication of the adenoviral vector is detected. Alternatively, thesample can be contacted with an adenovirus in which a reporter gene isunder control of a target cell-specific TRE. When such an adenovirus isintroduced into a biological sample, expression of the reporter geneindicates the presence of cells that allow a target cell-specific TRE tofunction. Alternatively, an adenovirus can be constructed in which agene conditionally required for cell survival is placed under control ofa target cell-specific TRE. This gene may encode, for example,antibiotic resistance. Later the biological sample is treated with anantibiotic. The presence of surviving cells expressing antibioticresistance indicates the presence of cells capable of targetcell-specific TRE function. A suitable biological sample is one in whichcells that allow a target cell-specific TRE to function, such as cancercells, may be or are suspected to be present. Generally, in mammals, asuitable clinical sample is one in which cancerous cells that allow atarget cell-specific TRE to function, such as carcinoma cells, aresuspected to be present. Such cells can be obtained, for example, byneedle biopsy or other surgical procedure. Cells to be contacted may betreated to promote assay conditions, such as selective enrichment,and/or solubilization. In these methods, cells that allow a targetcell-specific TRE to function can be detected using in vitro assays thatdetect adenoviral proliferation, which are standard in the art. Examplesof such standard assays include, but are not limited to, burst assays(which measure virus yield) and plaque assays (which measure infectiousparticles per cell). Propagation can also be detected by measuringspecific adenoviral DNA replication, which are also standard assays.

[0281] The invention also provides methods of modifying the genotype ofa target cell, comprising contacting the target cell with an adenovirusvector described herein, wherein the adenoviral vector enters the cell.

[0282] The invention further provides methods of suppressing tumor cellgrowth, preferably a tumor cell that allows a target cell-specific TREto function, comprising contacting a tumor cell with an adenoviralvector of the invention such that the adenoviral vector enters the tumorcell and exhibits selective cytotoxicity for the tumor cell. For thesemethods, the adenoviral vector may or may not be used in conjunctionwith other treatment modalities for tumor suppression, such aschemotherapeutic agents (such as those listed below), radiation and/orantibodies.

[0283] The invention also provides methods of lowering the levels of atumor cell marker in an individual, comprising administering to theindividual an adenoviral vector of the present invention, wherein theadenoviral vector is selectively cytotoxic toward cells that allow atarget cell-specific TRE to function. Tumor cell markers include, butare not limited to, CK-20. Methods of measuring the levels of a tumorcell marker are known to those of ordinary skill in the art and include,but are not limited to, immunological assays, such as enzyme-linkedimmunosorbent assay (ELISA), using antibodies specific for the tumorcell marker. In general, a biological sample is obtained from theindividual to be tested, and a suitable assay, such as an ELISA, isperformed on the biological sample. For these methods, the adenoviralvector may or may not be used in conjunction with other treatmentmodalities for tumor suppression, such as chemotherapeutic agents (suchas those listed below), radiation and/or antibodies.

[0284] The invention also provides methods of treatment, in which aneffective amount of an adenoviral vector(s) described herein isadministered to an individual. Treatment using an adenoviral vector(s)is indicated in individuals with cancer as described above. Alsoindicated are individuals who are considered to be at risk fordeveloping cancer (including single cells), such as those who have haddisease which has been resected and those who have had a family historyof cancer. Determination of suitability of administering adenoviralvector(s) of the invention will depend, inter alia, on assessableclinical parameters such as serological indications and histologicalexamination of tissue biopsies. Generally, a pharmaceutical compositioncomprising an adenoviral vector(s) in a pharmaceutically acceptableexcipient is administered. Pharmaceutical compositions are describedabove. For these methods, the adenoviral vector may or may not be usedin conjunction with other treatment modalities for tumor suppression,such as chemotherapeutic agents (such as those listed below), radiationand/or antibodies.

[0285] The amount of adenoviral vector(s) to be administered will dependon several factors, such as route of administration, the condition ofthe individual, the degree of aggressiveness of the disease, theparticular target cell-specific TRE employed, and the particular vectorconstruct (i.e., which adenovirus gene(s) is under target cell-specificTRE control) as well as whether the adenoviral vector is used inconjunction with other treatment modalities.

[0286] If administered as a packaged adenovirus, from about 10⁴ to about10¹⁴, preferably from about 10⁴ to about 10¹², more preferably fromabout 10⁴ to about 10¹⁰. If administered as a polynucleotide construct(i.e., not packaged as a virus), about 0.01 μg to about 100 μg can beadministered, preferably 0.1 μg to about 500 μg, more preferably about0.5 μg to about 200 μg. More than one adenoviral vector can beadministered, either simultaneously or sequentially. Administrations aretypically given periodically, while monitoring any response.Administration can be given, for example, intratumorally, intravenouslyor intraperitoneally.

[0287] The adenoviral vectors of the invention can be used alone or inconjunction with other active agents, such as chemotherapeutics, thatpromote the desired objective. Examples of chemotherapeutics which aresuitable for suppressing bladder tumor growth are BGC (bacillusCalmett-Guerin); mitomycin-C; cisplatin; thiotepa; doxorubicin;methotrexate; paclitaxel (TAXOL™); ifosfamide; gallium nitrate;gemcitabine; carboplatin; cyclosphasphamid; vinblastine; vincristin;fluorouracil; etoposide; bleomycin. Examples of combination therapiesinclude (CISCA (cyclophosphamide, doxorubicin, and cisplatin); CMV(cisplatin, methotrexate, vinblastine); MVMJ (methodtrextate,vinblastine, mitoxantrone, carboplain); CAP (cyclophosphamide,doxorubicin, cisplatin); MVAC (methotrexate, vinblastine, doxorubicin,cisplatin). Radiation may also be combined with chemotherapeuticagent(s), for example, radiation with cisplatin. Administration of thechemotherapeutic agents is generally intravesical (directly into thebladder) or intravenous.

[0288] The following examples are provided to illustrate but not limitthe invention.

EXAMPLES Example 1 Construction of a Replication-Competent AdenovirusVector Comprising an AFP-TRE and an EMCV IRES

[0289] The encephalomyocarditis virus (ECMV) IRES as depicted in Table 1was introduced between the E1A and E1B regions of areplication-competent adenovirus vector specific for cells expressingAFP as follows. Table 1 shows the 519 base pair IRES segment which wasPCR amplified from Novagen's pCITE vector by primers A/B as listed inTable 4. A 98 base pair deletion in the E1A promoter region was createdin PXC.1, a plasmid which contains the left-most 16 mu of Ad5. PlasmidpXC.1 (McKinnon (1982) Gene 19:33-42) contains the wild-type left-handend of Ad5, from Adenovirus 5 nt 22 to 5790 including the invertedterminal repeat, the packaging sequence, and the E1a and E1b genes invector pBR322. pBHG10 (Bett. et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806; Microbix Biosystems Inc., Toronto) provides the right-handend of Ad5, with a deletion in E3. The resultant plasmid, CP306(PCT/US98/16312), was used as the backbone in overlap PCR to generateCP624. To place a Sal1 site between E1a and E1b, primers C/D, E/F (Table4) were used to amplify CP306, plasmid derived from pXC.1 and lackingthe E1a promoter. After first round PCR using CP306 as template andprimers C/D, E/F, the resultant two DNA fragments were mixed togetherfor another round of overlapping PCR with primers C/F. The overlap PCRproduct was cloned by blunt end ligation to vector. The resultantplasmid, CP624 (Table 5), contains 100 bp deletion in E1a/E1b intergenicregion and introduces Sal1 site into the junction. On this plasmid, theendogenous E1a promoter is deleted, and the E1a polyadenylation signaland the E1b promoter are replaced by the Sal1 site. Next, the Sal1fragment of CP625 was cloned into the Sal1site in CP624 to generateCP627 (Table 5). CP627 has an EMCV IRES connecting adenovirus essentialgenes E1a and E1b. In CP627, a series of different tumor-specificpromoters can be placed at the PinA1 site in front of E1a to achievetranscriptional control on E1 expression. TABLE 4 Primer Sequence NoteA. 5′-GACGTCGACTAATTCCGGTTATTTTCCA For PCR EMCV IRES, GTCGAC is a SalIsite. B. 5′-GACGTCGACATCGTGTTTTTCAAAGGAA For PCR EMCV IRES, GTCGAC is aSalI site. C. 5′-CCTGAGACGCCCGACATCACCTGTG Ad5 sequence to 1314 to 1338.D. 5′-GTCGACCATTCAGCAAACAAAGGCGTTAAC Antisense of Ad5 sequence 1572 to1586. GTCGAC SalI site. Underline region overlaps with E. E.5′-TGCTGAATGGTCGACATGGAGGCTTGGGAG Ad5 sequence 1714 to 1728. GTCGAC is aSalI site. Underline region overlaps with D. F.5′-CACAAACCGCTCTCCACAGATGCATG Antisense of Ad5 sequence 2070 to 2094.

[0290] For generating a liver cancer-specific virus, an about 0.8 kb AFPpromoter fragment as shown in Table 3 was placed into the PinA1 site ofCP627 thereby yielding plasmid CP686. Full-length viral genomes wereobtained by recombination between CP686 and a plasmid containing a rightarm of an adenovirus genome. The right arms used in virus recombinationwere pBHGE3 (Microbix Biosystems Inc.), containing an intact E3 region,and pBHG11 or pBHG10 (Bett et al. (1994) containing a deletion in the E3region.

[0291] The virus obtained by recombination of CP686 with a right armcontaining an intact E3 region was named CV890. The virus obtained byrecombination of CP686 with a right arm containing a deleted E3 region(pBHG 10) was named CV840. The structure of all viral genomes wasconfirmed by conducting PCR amplifications that were diagnostic for thecorresponding specific regions.

[0292] Therefore, adenovirus vector designated CV890 comprises 0.8 kbAFP promoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B adeletion of the E1B promoter and an intact E3 region. Adenovirus vectorCV840 comprises AFP promoter, E1A, a deletion of the E1A promoter, EMCVIRES, E1B, a deletion of the E1B promoter and a deleted E3 region. TABLE5 Plasmid designation Brief description CP306 An E1A promoter deletedplasmid derived from pXC.1 CP624 Overlap PCR product from CP306 togenerate 100 bp deletion and introduce a Sal1 site at E1A and E1Bjunction; E1A and E1B promoter deleted in E1A/E1B intergenic region.CP625 EMCV IRES element ligated to PCR-blunt vector (Invitrogen pCR ®blunt vector). CP627 IRES element derived from CP625 by Sal1 digestionand ligated to CP624 Sal1 site placing IRES upstream from E1B. CP628Probasin promoter derived from CP251 by PinA1 digestion and cloned intoPinA1 site on CP627. CP629 HCMV IE promoter amplified from pCMV beta(Clontech) with PinA1 at 5′ and 3′ ends ligated into CP627 PinA1 site.CP630 A 163 bp long VEGF IRES fragment (Table 1) cloned into the Sal1site on CP628. CP686 AFP promoter from CP219 digested with PinA1 andcloned into PinA1 site on CP627.

Example 2 Construction of a Replication-Competent Adenovirus Vector Witha Probasin TRE and an EMCV IRES

[0293] The probasin promoter as shown in Table 3 was inserted at thePinAI site of plasmid CP627 (see Example 1) to generate CP628, whichcontains a probasin promoter upstream of E1A and an EMCV IRES betweenE1A and E1B. Full-length viral genomes were obtained by recombinationbetween CP628 and a plasmid containing a right arm of an adenovirusgenome. The right arms used in virus recombination were pBHGE3,containing an intact E3 region, and pBHG11 or pBHG10 containing adeletion in the E3 region. The structure of all viral genomes wasconfirmed by conducting PCR amplifications that were diagnostic for thecorresponding specific regions.

[0294] Therefore, adenovirus designated CV 834 comprises probasinpromoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B, adeletion of the E1B endogenous promoter and a deleted E3 region.

Example 3 Construction of a Replication-Competent Adenovirus Vector Witha hCMV-TRE and an EMCV IRES

[0295] The hCMV immediate early gene (IE) promoter from plasmid CP629,originally derived from pCMVBeta (Clonetech, Palo Alto) was inserted atthe PinAI site of plasmid CP627 (see Example 1) to generate CP629,containing a CMV IE promoter upstream of E1A and an IRES between E1A andE1B. Full-length viral genomes were obtained by recombination betweenCP629 and a plasmid containing a right arm of an adenovirus genome. Theright arms used in virus recombination were pBHGE3, containing an intactE3 region, and pBHG11 or pBHG10 containing a deletion in the E3 region.The structure of all viral genomes was confirmed by conducting PCRamplifications that were diagnostic for the corresponding specificregions.

[0296] Therefore, adenovirus vector designated CV835 comprises hCMV-IEpromoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B a deletionin the E1B endogenous promoter and a deleted E3 region. CV835 lacks thehCMV enhancer and is therefore not tissue specific. By adding the hCMVIE enhancer sequence to CV835, the vector is made tissue specific.

Example 4 Replication of IRES-Containing Adenovirus Vectors WithDifferent TREs Controlling E1 Expression

[0297] The viral replication of adenovirus vectors comprising theprobasin promoter (CV836 and CV834) generally considered a weakpromoter, and the human cytomegalovirus immediate early gene (HCMV-IE)promoter (CV837 and CV835), generally considered a strong promoter, werecharacterized in the virus yield assay.

[0298] Probasin promoter containing adenovirus vectors (seePCT/US98/04132), CV836 and CV834, and HCMV-IE promoter containingadenovirus vectors, CV837 and CV835, were tested against a panel of celllines for viral replication (indicative of lethality) and specificity.Cell lines 293 (the producer line), LNCap and HepG2 were plated at0.5×10⁶ per well in 6 well tissue culture plates, incubated for 24 hoursat 37° C., then infected with CV836, CV834 or CV837 and CV835 at amultiplicity of infection (MOI) of 2 plaque forming units per cell(PFU/cell) for 4 hours at 37° C. At the end of the infection period, themedium was replaced and the cells were incubated at 37° C. for a further72 hours before harvesting for a viral yield assay as described in Yu etal. (1999) Cancer Res. 59:1498-1504. The results are shown in FIG. 3.

[0299] The data demonstrate that the presence of an IRES element in theintergenic region between E1A and E1B does not significantly affectviral replication, as compared to control viruses lacking an IRES, suchas a wild-type AD5 with a deletion in the E3 region. In CV834, the lossof tissue cytotoxicity could be caused by the weakness of the probasinpromoter in the virus structure.

Example 5 Comparison of Dual TRE Vectors With Single TRE/IRES-ContainingVectors

[0300] Two liver cancer-specific adenovirus vectors, CV790 and CV733(also designated CN790 and CN733, respectively), were generated andcharacterized. See PCT/US98/04084. These viruses contain two AFP TREs,one upstrean of E1A and one upstream of E1B. They differ in that CV790contains an intact E3 region, while the E3 region is deleted in CV733.Replication of these two viruses was compared with that of the newlygenerated IRES-containing viruses, CV890 and CV840 (see Example 1).

[0301] Virus replication was compared, in different cell types, using avirus yield assay as described in Example 4. Cells were infected witheach type of virus and, 72 hrs after infection, virus yield wasdetermined by a plaque assay. FIGS. 4A and 4B show viral yield fordifferent viruses in different cell types. The results indicate thatvectors containing an IRES between E1A and E1B (CV890 and CV840), inwhich E1B translation is regulated by the IRES, replicate to similarextents as normal adenovirus and viruses with dual AFP TREs, inAFP-producing cells such as 293 cells and hepatoma cells. In SK-Hep-1(liver cells), PA-1 (ovarian carcinoma) and LNCaP cells (prostate cells)the IRES-containing viruses do not replicate as well as dual TRE orwild-type adenoviruses, indicating that the IRES-containing viruses havehigher specificity for hepatoma cells. Based on these results, it isconcluded that IRES-containing vectors have unaltered replicationlevels, but are more stable and have better target cell specificity,compared to dual-TRE vectors.

Example 6 Uroplakin Adenoviral Constructs Containing an EMCV IRES

[0302] A number of E3-containing viral constructs were prepared whichcontained uroplakin II sequences (mouse and/or human) as well as an EMCVinternal ribosome entry site (IRES). The viral constructs are summarizedin Table 6. All of these vectors lacked an E1A promoter and retained theE1A enhancer.

[0303] The 519 base pair EMCV]RES segment was PCR amplified fromNovagen's pCITE vector by primers A/B: primer A:5′-GACGTCGACTAATTCCGGTTATTTTCCA primer B 5′-GACGTCGACATCGTGTTTTTCAAAGGAA(GTCGAC is a SalI site).

[0304] The EMCV IRES element was ligated to PCR blunt vector (InvitrogenpCR® blunt vector).

[0305] CP1066

[0306] The 1.9 kb-(−1885 to +1) fragment of mouse UPII from CP620 wasdigested with AflIII (blunted) and HindIII and inserted into pGL3-Basicfrom CP620 which had been digested with XhoI (blunted) and HindIII togenerate CP1066.

[0307] CP1086

[0308] The 1.9 kb mouse UPII insert was digested with PinAI and ligatedwith CP269 (CMV driving E1A and IRES driving E1B with the deletions ofE1A/E1B endogenous promoter) which was similarly cut by PinAI.

[0309] CP1087

[0310] The 1 kb (−1128 to +1) human UPII was digested with PinAI fromCP665 and inserted into CP629 which had been cut by PinAI and purified(to elute CMV).

[0311] CP1088

[0312] The 2.2 kb (−2225 to +1) human UPII was amplified from CP657 withprimer 127.2.1 (5′-AGGACCGGTCACTATAGGGCACGCGTGGT-3′) PLUS 127.2.2(5′-AGGACCGGTGGGATGCTGGGCTGGGAGGTGG-3′) and digested with PinAI andligated with CP629 cut with PinAI.

[0313] CP627 is an Ad5 plasmid with an internal ribosome entry site(IRES) from encephelomycarditis virus (EMCV) at the junction of E1A andE1B. First, CP306 (Yu et al., 1999) was amplified with primer pairs96.74.3/96.74.6 and 96.74.4/96.74.5.

[0314] The two PCR products were mixed and amplified with primer pairs96.74.3 and 96.74.5. The resultant PCR product contains a 100 bpdeletion in E1A-E1B intergenic region and a new SaII site at thejunction. EMCV IRES fragment was amplified from pCITE-3a(+) (Novagen)using primers 96.74.1 and 96.74.2. The Sail fragment containing IRES wasplaced into Sail site to generate CP627 with the bicistronicE1A-IRES-E1B cassette. CP629 is a plasmid with CMV promoter amplifiedfrom pCMVbeta (Clontech) with primer 99.120.1 and 99.120.2 and clonedinto PinAI site of CP627.

[0315] CP657 is a plasmid with 2.2 kb 5′ flanking region of human UP IIgene in pGL3-Basic (Promega). The 2.2 kb hUPII was amplified by PCR fromGenomeWalker product with primer 100.113.1 and 100.113.2 and TA-clonedinto pGEM-T to generate CP655.

[0316] The 2.2 kb insert digested from SacII (blunt-ended) and KpnI wascloned into pGL3-Basic at HindIII (blunted) and KpnI to create CP657.

[0317] CP1089

[0318] The 1 kb (−965 to +1) mouse UPII was digested by PinAI from CP263and inserted into CN422 (PSE driving E1A and GKE driving E1B with thedeletions of E1A/E1B endogenous promoter) cut by PinAI and purified andfurther digested with EagI and ligated with 1 kb (−1128 to +1) humanUPII cut from CP669 with EagI.

[0319] CP1129

[0320] The 1.8 kb hUPII fragment with PinAI site was amplified fromCP657 with primer 127.50.1 and 127.2.2 and cloned into PinAI site ofCP629.

[0321] CP1131

[0322] CP686 was constructed by replacing the CMV promoter in CP629 withan AFP fragment from CP219. A 1.4 kb DNA fragment was released fromCP686 by digesting it with BssHII, filling with Klenow, then digestingwith BglII. This DNA fragment was then cloned into a similarly cut CP686to generate CP1199. In CP1199, most of the E1B 19-KDa region wasdeleted. The 1.8 kb hUPII fragment with PinAI site was amplified fromCP657 by PCR with primer 127.50.1 and 127.2.2 and inserted intosimilarly digested CP1199 to create CP1131.

[0323] The plasmids above were all co-transfected with pBHGE3 togenerate CV874 (from CP1086), CV875 (from CP1087), CV876 (from 1088) andCV877 (from CP1089), CV882 (from CP1129) and CV884 (from CP1131).CP1088, CP1129 and CP 1131 were cotransfected with pBHGE3 forconstruction of CV876, CV892 and CV884, respectively by lipofectAMINE(Gibco/BRL) for 11-14 days. pBHGE3 was purchased from Microbix, Inc.,and was described previously. The cells were lysed by three freeze-thawcycles and plaqued on 293 cells for a week. The single plaques werepicked and amplified by infection in 293 cells for 3-5 days. The viralDNAs were isolated from the lysates and the constructs were confirmed byPCR with primer 31.166.1/51.176 for CV876 and primer 127.50.1/51.176 forCV882 and CV884 at E1 region and primer 32.32.1/2 for all three virusesat E3 region. TABLE 6 Name Vector Ad 5 Vector E1A TRE E1B TRE E3 CV874CP1086 pBHGE3 1.9 kb mUPII IRES intact CV875 CP1087 pBHGE3 1.0 kb hUPIIIRES intact CV876 CP1088 pBHGE3 2.2 kb hUPII IRES intact CV877 CP1089pBHGE3 1.0 kb mUPII 1.0 kb hUPII intact (E1B promoter deleted) CV882CP1129 pBHGE3 1.8 kb hUPII IRES intact CV884 CP1131 pBHGE3 1.8 kb hUPiiIRES (E1B intact 19-kDa deleted)

[0324] Viruses are tested and characterized as described above.

[0325] Primer Sequences: 96.74.1 GACGTCGACATCGTGTTTTTCAAAGGAA 96.74.2GACGTCGACTAATTCCGGTTATTTTCCA 96.74.3 CCTGAGACGCCCGACATCACCTGTG 96.74.4TGCTGAATGGTCGACATGGAGGCTTGGGAG 96.74.5 CACAACCGCTCTCCACAGATGCATG 96.74.6GTCGACCATTCAGCAAACAAAGGCGTTAAC 100.113.1 AGGGGTACCCACTATAGGGCACGCGTGGT100.113.2 ACCCAAGCTTGGGATGCTGGGCTGGGAGGTGG 127.2.2AGGACCGGTGGGATGCTGGGCTGGGAGGTGG 127.50.1 AGGACCGGTCAGGCTTCACCCCAGACCCAC31.166.1 TGCGCCGGTGTACACAGGAAGTGA 32.32.1 GAGTTTGTGCCATCGGTCTAC 32.32.2AATCAATCCTTAGTCCTCCTG 51.176 GCAGAAAAATCTTCCAAACACTCCC 99.120.1ACGTACACCGGTCGTTACATAACTTAC 99.120.2 CTAGCAACCGGTCGGTTCACTAAACG

Example 7 Construction of a Replication-Competent Adenovirus Vector Witha Tyrosinase TRE and EMCV IRES

[0326] CP621 is a plasmid containing a human tyrosinase enhancer andpromoter elements in a PinAI fragment. This fragment is ligated to thePinAI site on CP627 to generate CP1078. CP1078 is combined with pBHGE3to generate a new melanoma specific virus, CV859. Table 3 depicts thepolynucleotide sequence of the PinAI fragment which contains atyrosinase promoter and enhancer.

Example 8 Construction of a Replication-Competent Adenovirus Vector Witha Probasin-TRE and a VEGF IRES

[0327] Using a strategy similar to that described in Example 1, the IRESfragment from the mouse vascular endothelial growth factor (VEGF) geneis amplified and cloned into CP628 at the SalI site. Table 1 depicts theIRES fragment obtainable from vascular endothelial growth factor (VEGF)mRNA. In order to clone this fragment into the E1a/E1b intergenicregion, two pieces of long oligonucleotide are synthesized. The senseoligonucleotide is shown in the Table, whereas the second piece is thecorresponding antisense one. After annealing the two together to createa duplex, the duplex is subjected to SalI digestion and the resultingfragment is cloned into the SalI site on CP628. The resulting plasmid,CP630, has a probasin promoter in front of E1a and an VEGF IRES elementin front of E1b. This plasmid is used to construct a prostatecancer-specific virus comprising the VEGF IRES element.

Example 9 Construction of a Replication-Competent Adenovirus Vector Withan AFP-TRE and a VEGF IRES

[0328] Using a strategy similar to Example 1, a PinAI fragment whichcontains AFP TRE can be obtained. This AFP TRE is cloned into the PinAIsite in front of E1A on CP628 yielding plasmid CP1077. This plasmid hasthe AFP TRE for E1 transcriptional control and the VEGF IRES elementbefore E1b. CP1077 can be recombined with pBHGE3 to generate aliver-specific adenovirus, designated as CV858.

Example 10 Construction of a Replication-Competent Adenovirus VectorWith a hKLK2-TRE and a EMCV IRES

[0329] Using a strategy similar to Example 1, the TRE fragment fromhuman glandular kallikrein II as shown in Table 3 was cloned into thePinAI site in CP627. The resultant plasmid, CP1079, is cotransfectedwith pBHGE3 to create CV860.

Example 11 Treatment of Hep3B Tumor Xenografts WithReplication-Competent Hepatoma Specific CV790 and Doxorubicin andHepatoma Specific CV890 and Doxorubicin

[0330] CV790 is an AFP producing hepatocellular carcinoma specificadenovirus, with E1A and E1B under the control of an identical AFPpromoter and enhancer (822 base pair promoter shown in Table 3) with anE3 region. The CV890 adenovirus construct is also a hepatoma orliver-specific adenoviral mutant with the E1A and E1B genes undertranscriptional control of 822 bp AFP promoter (827 bp includingnucleotides for restriction site), wherein E1B is under translationalcontrol of EMCV IRES and having an intact E3 region. The structure ofCV890 therefore reads as AFP/E1A, IRES/E1B, E2, E3, E4. In vivo studiesof the efficacy of combinations of CV790 and doxorubicin and CV890 anddoxorubicin were performed according to the protocols described indetail in Example 4, with minor alterations which are described below.

[0331] Xenografts in the study of CV790 and CV890 combined withchemotherapeutic agents utilized liver carcinoma Hep3B cells. Virus,CV790 or CV890, was administered by a single intravenous injection of1×10 1 particles through the tail veins of the nude mice. One day aftervirus delivery, a single dose of doxorubicin was given to each animal byi.p. injection. The doxorubicin dose was 10 mg/kg for both doxorubicinalone and doxorubicin combined with virus treatments. Tumor volume wasmeasured once a week for six weeks. Tumors were measured weekly in twodimensions by external caliper and volume was estimated by the formula[length (mm)×width (mm)₂]/2.

[0332]FIG. 8 depicts the anti-tumor activity of CV890 containing an IRESas compared to CV 790 containing dual TREs. As FIG. 8 demonstrates,relative tumor volume was less with administration of CV890 thanadministration of CV790.

[0333] Furthermore, both CV790/doxorubicin and CV890/doxorubicintreatment of the hepatoma showed synergistic results. Four days aftertreatment with either CV790/doxorubicin or CV890/doxorubicin therelative tumor volume was less than 10%. Unlike mice treated with eithervirus alone or doxorubicin alone, after day 4, the relative tumor volumedid not increase for either the either CV790/doxorubicin orCV890/doxorubicin treated mice. At day 6 in the control mice, therelative tumor volume was approximately 1000% in the CV790 study andapproximately 600% in the CV890 study. The relative tumor volumes ofmice treated with virus alone were 250% (CV790) and 520% (CV890) whilethe relative tumor volumes for mice treated with doxorubicin alone were450% with 280% in the CV790 study and 500% in the CV890 study.

Example 12 In Vitro Characterization of Melanocyte-SpecificTRE-Containing Adenoviral Constructs

[0334] An especially useful objective in the development of melanocytecell-specific adenoviral vectors is to treat patients with melanoma.Methods are described below for measuring the activity of amelanocyte-specific TRE and thus for determining whether a given cellallows a melanocyte-specific TRE to function.

[0335] Cells and Culture Methods

[0336] Host cells such as, HepG2 (liver); Lovo (colon); LNCaP(prostate); PMEL (melanoma); SKMel (melanoma); G361 (melanoma) and MeWocells are obtained at passage 9 from the American Type CultureCollection (Rockville, Md.). MeWo cells are maintained in RPMI 1640medium (RPMI) supplemented with 10% fetal bovine serum (FBS; IntergenCorp.), 100 units/mL of penicillin, and 100 units/mL streptomycin. MeWocells being assayed for luciferase expression are maintained in 10%strip-serum (charcoal/dextran treated fetal bovine serum to remove T3,T4, and steroids; Gemini Bioproduct, Inc., Calabasas, Calif.) RPMI.

[0337] Transfections of MeWo Cells

[0338] For transfections, MeWo cells are plated out at a cell density of5×10⁵ cells per 6-cm culture dish (Falcon, N.J.) in complete RPMI. DNAsare introduced into MeWo cells after being complexed with a 1:1 molarlipid mixture of N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammoniumchloride (DOTAP™; Avanti Polar Lipids, Ala.) anddioleoyl-phosphatidylethanolamine (DOPE™; Avanti Polar Lipids, Ala.);DNA/lipid complexes are prepared in serum-free RPMI at a 2:1 molarratio. Typically, 8 μg (24.2 nmole) of DNA is diluted into 200 μL ofincomplete RPMI and added dropwise to 50 mmole of transfecting, lipidsin 200 μL of RPMI with gentle vortexing to insure homogenous mixing ofcomponents. The DNA/lipid complexes are allowed to anneal at roomtemperature for 15 minutes prior to their addition to MeWo cells. Mediumis removed from MeWo cells and replaced with 1 mL of serum-free RPMIfollowed by the dropwise addition of DNA/lipid complexes. Cells areincubated with complexes for 4-5 hours at 37° C., 5% CO₂. Medium wasremoved and cells washed once with PBS. The cells were then trypsinizedand resuspended in 10% strip-serum RPMI (phenol red free). Cells werereplated into an opaque 96-well tissue culture plate (Falcon, N.J.) at acell density of 40,000 cells/well per 100 μL media and assayed.

[0339] Plaque Assays

[0340] To determine whether the adenoviral constructs described abovereplicate preferentially in melanocytes, plaque assays are performed.Plaquing efficiency is evaluated in the following cell types: melanomacells (MeWo), prostate tumor cell lines (LNCaP), breast normal cell line(HBL-100), ovarian tumor cell line (OVCAR-3, SK-OV-3), and humanembryonic kidney cells (293). 293 cells serve as a positive control forplaquing efficiency, since this cell line expresses Ad5 E1A and E1Bproteins. For analyzing constructs comprising a melanocyte-specific TRE,cells that allow a melanocyte-specific TRE to function, such as the celllines provided above and cells that do not allow such function, such asHuH7, HeLa, PA-1, or G361, are used. The plaque assay is performed asfollows: Confluent cell monolayers are seeded in 6-well dishes eighteenhours before infection. The monolayers are infected with 10-fold serialdilutions of each virus. After infecting monolayers for four hours inserum-free media (MEM), the medium is removed and replaced with asolution of 0.75% low melting point agarose and tissue culture media.Plaques are scored two weeks after infection.

Example 13 Construction of a Replication-Competent Adenovirus VectorWith a CEA-TRE and a EMCV IRES

[0341] Using a strategy similar to Example 1, the TRE fragment fromCarcinembryonic antigen (CEA)(Table 3, SEQ ID NO:14) is used toconstruct virus designated CV873. A PinAI fragment containing theCEA-TRE was cloned into the PinAI site in front of E1A CP627 for thetranscriptional control. The resultant plasmid CP1080 is used togetherwith pBHGE3 to generate CV873.

Example 14 In Vitro and In Vivo Assays of Anti-Tumor Activity

[0342] An especially useful objective in the development of urothelialcell-specific adenoviral vectors is to treat patients with bladdercancer. An initial indicator of the feasibility is to test the vector(s)for cytotoxic activity against cell lines and tumor xenografts grownsubcutaneously in Balb/c nu/nu mice.

[0343] In Vitro Characterization of CV876

[0344] Virus Yield Assay for CV876

[0345] 5×10⁵293, RT-4, SW780, PA-1, G361, MKNI, HBL-100, Fibroblast(from lung) and Smooth muscle cells (from bladder) were plated into eachwell of six-well plates. Twenty-four hours later, medium was aspiratedand replaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5with E3) or CV876 at a MOI of 2 pfu/cell. After a 4-h incubation at 37°C., cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. After an additional 72 h at 37° C., the cellswere scraped into medium and lysed by three freeze-thaw cycles. Thelysates were tested for virus production by triplicate plaque assay for8-10 days under semisolid agarose on 293 cells.

[0346] Unlike wt. Ad5, CV802 which grows well in all of the cellstested, CV876 replicates much better in permissive cells (293, RT-4 andSW780) than in non-permissive cells (PA-1, G361, MKN1, HBL-100 andprimary cells) by about 100-10000 fold. Noticeably, the replication inSW780 for CV876 is about 100 fold less than CV802, which indicates thelimitation of this virus in efficacy.

[0347] Growth Curve Experiment for CV876

[0348] 5×10⁵ RT-4, PA-1, Smooth muscle and Fibroblast cells were platedinto each well of six-well plates. Twenty-four hours later, medium wasaspirated and replaced with 1 ml of serum-free RPMI 1640 containingCV802 (wt.Ad5 with 133) or CV876 at a MOI of 2 pfu/cell. After a 4-hincubation at 37° C., cells were washed with prewarmed PBS, and 2 ml ofcomplete RPMI 1640 were added to each well. At different time points of0, 12, 24, 36, 48, 72, 96 and 120 h, the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

[0349] Very similar as in virus yield assay, CV876 replicates well onlyin RT-4 but not in primary cells and PA-1 over a 120 h period of time.However, CV876 does show a delay of replication in RT-4 compared toCV802.

[0350] Cytopathic Effect Assay for CV876

[0351] 5×10⁵293, RT-4, SW780, PA-1, MKN1 and LNCap were plated into eachwell of six-well plates. Twenty-four hours later, medium was aspiratedand replaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5with E3) or CV876 at increasing MOI from 0.001 to 10 (the data shown wasat MOI 1). After a 4-h incubation at 37° C., medium was replaced with 3ml of complete RPMI 1640 and incubated at 37° C. for 6-8 days whencytopathic effect was observed for CV802 at MOI 0.01.

[0352] CV802 shows efficacy in all the cells tested while CV876 onlykills the permissive cells (293, RT-4 and SW780) but not thenon-permissive cells (PA-1, MKN-1 and LNCap).

[0353] MTT Assay for CV876

[0354] 2×10⁴293, RT-4, SW780, MKN1, PA-1, HBL-100, Smooth muscle cells(from bladder) and Fibroblast (from lung) were plated into each well of96-well plates. Twenty-four hours later, the cells were infected withCV802 and CV876 at increasing MOI from 0.001 to 10 in complete RPMI1640. A rapid colorimetric assay for cell growth and survival was run atdifferent time point of day 1, 3, 5, 7 and 10. The medium was replacedby 50 ul of MTT at 1 mg/ml solution, which is converted to an insolublepurple formazan by dehydrogenase enzymes present in active mitochondriaof live cells. After 3-4 h incubation at 37° C., the solution wasreplaced by isopropanol and the plates were incubated at 30° C. for 1 hand read at 560 nm test wavelength and 690 nm reference wavelength.

[0355] Similar as the results in CPE assay, CV876 shows efficacy only inpermissive cells but not in non-permissive cells. Again, in RT-4 andSW780, CV876 kills the cells much slower than CV802.

[0356] In Vitro Characterization of CV882

[0357] Virus Yield Assay for CV882

[0358] 5×10⁵293, RT-4, SW780, G361, LNCap, HBL-100, MKN1, PA-1,Fibroblast and Smooth muscle cells were plated into each well ofsix-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV882 at a MOI of 2 pfu/cell. After a 4-h incubation at 37° C.,cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. After an additional 72 h at 37° C., the cellswere scraped into medium and lysed by three freeze-thaw cycles. Thelysates were tested for virus production by triplicate plaque assay for8-10 days under semisolid agarose on 293 cells.

[0359] The replication of CV882 in permissive cells (293, RT-4 andSW780) is comparable to CV802 (the difference is less than 100 fold)while it shows over 1000-1000000 fold difference in non-permissive cells(G361, LNCap, HBL-100, MKN1, PA-1 and primary cells).

[0360] Growth Curve Experiment for CV882

[0361] 5×10⁵RT-4, PA-1, and Fibroblast cells were plated into each wellof six-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV882 at a MOI of 2 pfu/cell. After a 4-h incubation at 37° C.,cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. At different time points of 0, 12, 24, 36, 48,72, 96 and 120 h, the cells were scraped into medium and lysed by three,freeze-thaw cycles. The lysates were tested for virus production bytriplicate plaque assay for 8-10 days under semisolid agarose on 293cells.

[0362] Very similar as in virus yield assay, CV882 replicates well onlyin RT-4 but not in primary cells and PA-1 over a 120 h period of time.Additionally, CV882 shows better replication in RT-4 compared to CV876.

[0363] Cytopathic Effect Assay for CV882

[0364] 5×10⁵293, RT-4, SW780, HBL-100, G361, PA-1 and Fibroblast cellswere plated into each well of six-well plates. Twenty-four hours later,medium was aspirated and replaced with 1 ml of serum-free RPNI 1640containing CV802 (wt.Ad5 with E3) or CV882 at increasing MOI from 0.001to 10 (the data shown was at MOI 1). After a 4-h incubation at 37° C.,medium was replaced with 3 ml of complete RPMI 1640 and incubated at 37°C. for 6-8 days when cytopathic effect was observed for CV802 at MOI0.01.

[0365] CV802 shows efficacy in all the cells tested while CV882 onlykills the permissive cells (293, RT-4 and SW780) but not thenon-permissive cells (HBL-100, G361, PA-1 and Fibroblast cells).

[0366] MTT Assay for CV882

[0367] 2×10⁴RT-4, SW780, PA-1, HBL-100, U118 and Fibroblast were platedinto each well of 96-well plates. Twenty-four hours later, the cellswere infected with CV802 and CV882 at increasing MOI from 0.001 to 10 incomplete RPMI 1640. A rapid colorimetric assay for cell growth andsurvival was run at different time points of day 1, 3, 5, 7 and 10. Themedium was replaced by 50 ul of MTT at 1 mg/ml solution, which isconverted to an insoluble purple formazan by dehydrogenase enzymespresent in active mitochondria of live cells. After 3-4 h incubation at37° C., the solution was replaced by isopropanol and the plates wereincubated at 30° C. for 1 h and read at 560 nm test wavelength and 690nm reference wavelength.

[0368] Similar as the results in CPE assay, CV882 shows efficacy only inpermissive cells but not in non-permissive cells.

[0369] In Vitro Characterization of CV884

[0370] Virus Yield Assay for CV884

[0371] 5×10⁵293, RT-4, SW780, G361, LNCap, HBL-100, MKN1, PA-1,Fibroblast and Smooth muscle cells were plated into each well ofsix-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV984 at a MOI of 2 pfu/cell. After a 4-h incubation at 37° C.,cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. After an additional 72 h at 37° C., the cellswere scraped into medium and lysed by three freeze-thaw cycles. Thelysates were tested for virus production by triplicate plaque assay for8-10 days under semisolid agarose on 293 cells.

[0372] The replication of CV884 is very similar as CV802 in permissivecells (293, RT-4 and SW780) but shows over 1000 fold difference withCV802 in non-permissive cells (G361, LNCap, HBL-100, MKN1, PA-1 andprimary cells). CV884 shows better efficacy than CV876 and CV882 withoutlosing much specificity.

[0373] Growth Curve Experiment for CV884

[0374] 5×10⁵RT-4, PA-1, Smooth muscle and Fibroblast cells were platedinto each well of six-well plates. Twenty-four hours later, medium wasaspirated and replaced with 1 ml of serum-free RPMI 1640 containingCV802 (wt.Ad5 with E3) or CV884 at a MOI of 2 pfu/cell. After a 4-hincubation at 37° C., cells were washed with prewarmed PBS, and 2 ml ofcomplete RPMI 1640 were added to each well. At different time points of0, 12, 24, 36, 48, 72, 96 and 120 h, the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

[0375] Very similar as in virus yield assay, CV884 replicates very wellonly in RT-4 (similar as CV802) but not in primary cells and PA-1.Again, the replication of CV884 is better than CV882 and CV876.

[0376] Cytopathic Effect Assay for CV884

[0377] 5×10⁵293, RT-4, SW780, G361, PA-1 and Fibroblast cells wereplated into each well of six-well plates. Twenty-four hours later,medium was aspirated and replaced with 1 ml of serum-free RPMI 1640containing CV802 (wt.Ad5 with E3) or CV884 at increasing MOI from 0.001to 10 (the data shown was at MOI 1). After a 4-h incubation at 37° C.,medium was replaced with 3 ml of complete RPMI 1640 and incubated at 37°C. for 6-8 days when cytopathic effect was observed for CV802 at MOI0.01.

[0378] CV802 shows efficacy in all the cells tested while CV884 onlykills the permissive cells (293, RT-4 and SW780) but not thenon-permissive cells (G361, PA-I and Fibroblast cells).

[0379] MTT Assay for CV884

[0380] 2×10⁴293, RT-4, SW780, U118, Fibroblast and Smooth muscle cellswere plated into each well of 96-well plates. Twenty-four hours later,the cells were infected with CV802 and CV884 at increasing MOI from0.001 to 10 in complete RPMI 1640. A rapid colorimetric assay for cellgrowth and survival was run at different time points of day 1, 3, 5, 7and 10. The medium was replaced by 50 ul of MTT at 1 mg/ml solutionwhich is converted to an insoluble purple formazan by dehydrogenaseenzymes present in active mitochondria of live cells. After 3-4 hincubation at 37° C., the solution was replaced by isopropanol and theplates were incubated at 30° C. for 1 h and read at 560 nm testwavelength and 690 nm reference wavelength.

[0381] Similar as the results in CPE assay, CV884 shows strong efficacy(similar as wt. Ad5) only in permissive cells but not in non-permissivecells.

[0382] In Vivo Activity of CV808

[0383] Mice were given subcutaneous (SC) injections of 1×10⁶ sW780cells. When tumors grew to about 500 mm³, CV808 was introduced into themice (5×10⁷ PFU of virus in 0.1 ml PBS and 10% glycerol) intratumorally.Control mice received vehicle alone. Tumor sizes were measured weekly.The results are shown in FIG. 11. The data indicate that CV808 waseffective at suppressing tumor growth.

[0384] While it is highly possible that a therapeutic based on theviruses described here would be given intralesionally (i.e., directinjection), it would also be desirable to determine if intravenous (IV)administration of adenovirus vector can affect tumor growth. If so, thenit is conceivable that the virus could be used to treat metastatic tumordeposits inaccessible to direct injection. For this experiment, groupsof mice bearing bladder epithelial tumors are inoculated with 108 to1010 PFU of an adenoviral vector by tail vein injection, or with bufferused to carry the virus as a negative control. The effect of IVinjection of the adenoviral vector on tumor size is compared to vehicletreatment.

Example 15 Synergistic Effect of CV 890 With Chemotherapeutics

[0385] Materials and Methods

[0386] Cells. Hepatocellular carcinoma cell lines HepG2, Hep3B,PLC/PRF/5, SNU449, and Sk-Hep-1, Chang liver cell (human normal livercells), as well as other tumor cell lines PA-1 (ovarian carcinoma),UM-UC-3 (bladder carcinoma), SW 780 (bladder carcinoma), HBL100 (breastepithelia), Colo 201 (Colon adenocarcinoma), U 118 MG (glioblastoma) andLNCaP (prostate carcinoma) were obtained from the American Type CultureCollection. HuH-7 (liver carcinoma) was a generous gift of Dr. PatriciaMarion (Stanford University). 293 cells (human embryonic kidneycontaining the E1 region of Adenovirus) were purchased from Microbix,Inc. (Toronto, Canada). The primary cells nBdSMC (normal human bladdersmooth muscle cells), nHLFC (normal human lung fibroblast cells), andnHMEC (normal human mammary epithelial cells) were purchased fromClonetics (San Diego, Calif.). All tumor cell lines were maintained inRPMI 1640 (BioWhittaker, Inc.) supplemented with 10% fetal bovine serum(Irvine Scientific), 100 U/ml penicillin and 100 ug/ml streptomycin.Primary cells were maintained in accordance with vendor instructions(Clonetics, San Diego). Cells were tested for the expression of AFP byimmunoassay (Genzyme Diagnostics, San Carlos, Calif.).

[0387] Virus yield and one-step growth curves. Six well dishes (Falcon)were seeded with 5×10⁵ cells per well of calls of interest 24 hrs priorto infection. Cells were infected at an multiplicity of infection (MOI)of 2 PFU/cell for three hours in serum-free media. After 3 hours, thevirus containing media was removed, monolayers were washed three timeswith PBS, and 4 ml of complete media (RPMI1640+10% FBS) was added toeach well. 72 hours post infection, cells were scraped into the culturemedium and lysed by three cycles of freeze-thaw.

[0388] The one-step growth curves time points were harvested at varioustime points after infection. Two independent infections of each viruscell-combination were titered in duplicate on 293 cells (Yu et al.,1999, Cancer Research, 59:1498-1504.

[0389] Northern blot analysis. Hep3B or HBL100 cells were infected at anMOI of 20 PFU/cell (plaque forming unit per cell) with either CV802 orCV890 and harvested 24 hours post infection. Total cell RNA was purifiedusing the RNeasy protocol (Qiagen). The Northern blot was conductedusing NorthernMax Plus reagents (Ambion, Austin, Tex.). 5 ug of RNA wasfractionated on a 1% agarose, formaldehyde-based denaturing gel andtransferred to a BrightStar-Plus (Ambion) positively charged membrane bycapillary transfer. The antisense RNA probes for E1A (adenovirus genome501 bp to 1141 bp) or E1B (1540 bp-3910 bp) were PCR products cloned inpGEM-T easy (Promega) and transcription labeled with [³²P] UTP. Blotswere hybridized at 68° C. for 14 hours with ZipHyb solution and washedusing standard methods (Ambion). Membranes were exposed to BioMax film(Kodak).

[0390] Western blot analysis. Hep3B or HBL100 cells were infected at MOIof 20 PFU/cell with either CV802 or CV890 and harvested 24 hours postinfection. Cells were washed with cold PBS and lysed for 30 min on icein (50 mM Tris, pH 8.0, 150 mM NaCl, 1% IGEPAL CA360 a NP40 equivalent(Sigma), 0.5% sodium deoxycholate, and protease inhibitor cocktail from(Roche, Palo Alto, Calif.). After 30 min centrifugation at 4 C, thesupernatant was harvested and the protein concentration determined withprotein assay ESL kit (Roche). Fifty micrograms of protein per lane wereseparated on 816% SDS-PAGE and electroblotted onto Hybond ECL membrane(Amersham Pharmacia, Piscataway, N.J.). The membrane was blockedovernight in PBST (PBS with 0.1% Tween-20) supplemented with 5% nonfatdry milk. Primary antibody incubation was done at room temperature for2-3 hrs with PBST/1% milk diluted antibody, followed by wash and 1 hrincubation with diluted horseradish peroxidase-conjugated secondaryantibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). Enhancedchemiluminescence (ECL; Amersham Pharmacia) was used for the detection.E1A antibody (clone M58) was from NeoMarkers (Fremont, Calif.), E1B-21kD antibody was from Oncogene (Cambridge, Mass.). All antibodies wereused according manufacturer's instruction.

[0391] Cell viability assay and statistical analysis. To determine thecell killing effect of virus and chemotherapeutic agent in combinationtreatment, a cell viability assay was conducted as previously describedwith modifications (Denizot, 1986, Journal Immunology. Methods,89:271-277). On 96 well plates, cells of interest were seeded at 10,000calls per well 48 hr prior to infection. Cells were then treated withvirus alone, drug alone, or in combination. Cell viability was measuredat different time points by removing the media, adding 50

l of 1 mg/ml solution of MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)(Sigma, St. Louis, Mo.) and incubating for 3 hrs at 37° C. Afterremoving the MTT solution, the crystals remaining in the wells weresolubilized by the addition of 50

l of isopropanol followed by 30 C incubation for 0.5 hr. The absorbencywas determined on a microplate reader (Molecular Dynamics) at 560 nm(test wavelength) and 690 nm (reference wavelength). The percentage ofsurviving cells was estimated by dividing the OD₅₅₀-OD₆₅₀ of virus ordrug treated cells by the OD₅₅₀-OD₆₅₀ of control cells. 6 replicasamples were taken for each time point and each experiment was repeatedat least three times.

[0392] For statistical analysis, CurveExpert (shareware by Daniel Hyams,version 1.34) was used to plot the dose-response curves for virus anddrugs. Based upon the dose-response curves, the isobolograms were madeaccording to the original theory of Steel and Peckham (1993, Int. J.Rad. Onc. Biol. Phys., 5:85) and method described in Aoe et al. (1999,Anticancer Res. 19:291-299).

[0393] Animal studies. Six to eight week old athymic BALB/C nu/nu micewere obtained from Simonson Laboratories (Gilroy, Calif.) and acclimatedto laboratory conditions one week prior to tumor implantation.Xenografts were established by injecting 1×10⁶ Hep3B, HepG2 or LNCAPcells suspended in 100

l of RPMI 1640 media subcutaneously. When tumors reached between 200 mm³and 300 mm³, mice were randomized and dosed with 100

l of test article via intratumoral or the tail vein injection. Tumorswere measured in two dimensions by external caliper and volume wasestimated by the formula [length (mm)×width (mm)²]/2. Animals werehumanely killed when their tumor burden became excessive. Serum washarvested weekly by retro-orbital bleed. The level of AFP in the serumwas determined by AFP Immunoassay kit (Genzyme Diagnostics, San Carlos,Calif.). The difference in mean tumor volume and mean serum AFPconcentration between treatment groups was compared for statisticalsignificance using the unpaired, two-tailed, t-test.

[0394] Transcription and Translation of E1A/E1B Bicistronic Cassette ofCV890 in Different Cells.

[0395] In wild type adenovirus infection, E1A and E1B genes produce afamily of alternatively spliced products. It has been found that thereare five E1A mRNAs, among them 12S (880 nucleotides, nts) and 13S (1018nts) mRNAs are the dominant ones that are expressed both early and lateafter infection. The 12S and 13S mRNAs encode the gene product of 243amino acids (243R) and 289 amino acids (289R) respectively (reviewed byShenk, 1996). The two major E1B transcripts that code for 19 kD and 55kD proteins are 12S (1031 nts) and 22S (2287 nts) mRNAs. E1B 12S mRNAonly codes the 19 kD product, whereas the 22S mRNA codes for both 19 kDand 55 kD products due to different initiation sites during translation.In the current study, the generation of E1A-IRES-E1B bicistroniccassette was expected to change the pattern of E1A and E1B transcriptsin viral infection. Therefore, Northern blot analysis was conducted toevaluate the steady-state level of E1A and E1B transcripts. First, CV802or CV890 were infected to Hep3B (AFP) or HBL100 (AFP) cells for 24hours. The total RNA samples were separated on agarose gels andprocessed for Northern blot by hybridizing to antisense RNA probes. TheNorthern blot with E1A probe visualized the 12S and 13S mRNAs in bothwild type CV802 infected cells. For CV890, E1A transcripts can only beseen in Hep3B cells, indicating the conditional transcription of E1A. Itis of interest to find that in CV890, there is only one large transcript(about 3.51 Kb), whereas the 12S and 13S mRNAs are no longer present.This large transcript indicates the continuous transcription ofE1A-IRES-E1B bicistronic cassette, suggesting an alteration of viral E1Asplicing pattern in CV890. Transcription of E1B from CV890 also appearsto be AFP-dependent. It is clear that both 12S and 22S mRNAs of E1B werepresent in wild type CV802 samples, whereas the 128 mRNA and an enlarged22S mRNA (3.5 Kb) appeared in CV890 infected cells. Obviously, theidentity of this enlarged mRNA is the same 3.5 Kb transcript asvisualized in E1A blot, which is from the transcription of E1A/E1Bbicistronic cassette. Therefore, the E1B mRNA is tagged after E1A mRNAin this large transcript. This large transcript contains all the codinginformation for E1A, E1B 19 kD and E1B 55 kD. The mRNA splice patternthat appears in CV802 is not valid in CV890, the 12S mRNA with E1B probedisappeared. Meanwhile, in the E1B Northern blot, due to the selectionof our E1B probe (1540 bp-3910 bp), mRNA of the Adenovirus gene IX (3580bp-4070 bp), the hexon-associated protein, was also detected. In CV890infected Hep3B cells, gene IX expression is equivalent to that of CV802,whereas in CV890 infected HBL100, its expression was also completelyshut down. This result further demonstrated that the AFP controlledE1A/E1B expression is the key for late gene expression as well as viralreplication.

[0396] Results of the same samples in the Western blot also indicatethat CV890 has AFP dependent expression of E1A and E1B. Under ourexperimental conditions, E1A expression level of CV890 in Hep3B cells issimilar to that of CV802. However, when E1B 19 kD protein was detected,it was found that the expression level was much lower than CV802 E1A.Previously, it has been addressed that IRES-mediated second gene hasless expression (Mizuguchi et al., 2000, Mol. Ther. 1:376-382). Takentogether, CV890 infection in permissive Hep3B cells can produce normalamounts of E1A and lesser amounts of E1B proteins capable of initiatinga normal productive infection. In AFP⁻ cells, however, this process wasattenuated due to a lack of E1A and E1B gene transcription andtranslation. These data demonstrated that the expression of both E1A andE1B genes are under the control of AFP TRE and the artificial E1A/E1Bbicistronic cassette is functioning properly in CV890.

[0397] In Vitro Replication Specificity of CV890 in Tumor Cells andPrimary Cells.

[0398] From in vitro comparison of virus yield, CV890 has a betterspecificity profile than CV732 (CV732 is an AFP-producing, cell-specificadenovirus variant in which the E1A gene is under control of AFP-TRE).In order to gain further insights of using CV890 in liver cancertherapy, more tumor cell lines and primary cells were tested tocharacterize in vitro virus replication. First, all cells in the studywere analyzed for their AFP status by AFP immune assay. Based on AFPproduced in the cells and media, all the cells were divided into threegroups, high (>2.5

g/10⁶ cells/10 days), low (<0.6

g/10⁶ cells/10 days) and none (undetectable in our study) (Table 7). Itwas confirmed that replication of CV890 in different cell linescorrelates well with the AFP status of the host cell. Among the group ofliver cell lines, CV890 only replicates well in AFP⁺ cells, includingHep3B, HepG2, Huh7, SNU449 and PLC/PRF/5. The amount of AFP required forthe promoter activity seems very low as one of the hepatoma cell lines,SNU449, a previous reported AFP⁻ cell (Park et al., 1995, Int. J. Cancer62:276-282), produces very low AFP (about 60 ng/10⁶ cells/10 days)compared to other cells. Nevertheless, even with very low amount of AFP,SNU449 cells can still support CV890 replication to the extentcomparable to cells producing significantly higher levels of AFP such asHepG2. Compared to CV802, CV890 is attenuated 5,000 to 100,000 fold incells that do not produce AFP, including the hepatoma cell Sk-Hep1 andChang liver cell, other tumor cells and primary cells. Taken togetherthe results indicate that CV890 has shown a good specificity profilefrom a broad spectrum of tumor cells. Among them, only the AFP⁺ livercells, AFP production level from high to low, are permissive for CV890.

[0399] In another experiment, CV890 was compared to CV802 for theirsingle step growth curves on different cells. Results demonstrated thatCV890 has a similar growth kinetics to wild-type CV802 in AFP⁺ cellsexcept that virus yields are slightly lower (2-8 fold) in low AFPproducing cells. In consideration of experimental error, there is nodramatic difference in the replication of CV890 and CV802 in AFP⁺hepatoma cells. However, the growth curves of CV890 in AFP⁻ cells showedclear attenuation. During a 5 day experiment, CV890 failed to replicatein AFP⁻ cells including hepatoma cell (Change liver) and primary cells(nHLFC). From all the in vitro virus replication studies, it is clearthat replication of CV890 is under the tight control of AFP-TRE and thisadenovirus variant has an excellent specificity profile ofpreferentially targeting AFP producing hepatocellular carcinoma cells.

[0400] In Vivo Specificity and Efficacy of CV890.

[0401] CV890 specificity was also evaluated in animals bearing prostatecancer LNCaP xenografts. In this in vivo test, nude mice with prostatexenograft were intravenously injected with either CV890 or CV787, aprostate cancer specific adenovirus variant (Yu et al., 1999, CancerResearch, 59:4200-4203). Tumor volumes were documented and indicatedthat only CV787 had a significant antitumor efficacy in LNCaPxenografts, while tumors in the animals treated with CV890 grew, from400 mm³ to approximately 1200 mm³ in six weeks, similar to the grouptreated with vehicle. This study indicates that CV890 does not attackLNCaP xenograft and keeps the good specificity profile under in vivoconditions.

[0402] To evaluate in vivo antitumor efficacy of CV890, differentstudies were carried out in the nude mouse model harboring humanhepatoma xenografts. First, BALB/c nu/nu mice with HepG2 or Hep3Bxenografts were established, animals were further challenged with singledose or multiple doses of CV890 into the tumor mass (intratumoraladministration, IT) or via their tail vein (intravenous administration,IV). Tumor volume and the level of serum AFP were monitored weekly afterthe start of treatment, and hence the efficacy of the treatment wasdetermined. The in vitro cytotoxicity study has demonstrated that CV890has a better cytolytic effect than CV732. In order to further examinetheir antitumor activity, we first conducted animal study to compareCV890 to CV732. Animals harboring 300 mm³ Hep3B xenograft were grouped(n=6) and injected with vehicle alone (control group), CV890 (1×10¹¹particles/dose, CV890 group), or CV732 (1×10¹¹ particles/dose, CV732group). The Hep3B xenograft is a very aggressive tumor model and tumorsgrow very fast. Most animals can not survive long because of excessivetumor burden. During a six week study, single intravenous administrationof CV890 have shown significant tumor growth inhibition, whereas controlmice had over 10 fold tumor growth at week 5. In the group treated withCV732, single dose IV injection also reduced the tumor growth ascompared to control group, however, it was much less effective comparedto CV890. For example, the average tumor volume of the CV890 treatedgroup dropped from 312 mm³ to 219 mm³, while tumor volume increased from308 mm³ to 1542 mm³ 5 weeks after treatment in control. Both controlgroup and the CV732 group were terminated at week 5 because excessivetumor size. Previously, CV732 has been demonstrated to restrict thehepatoma tumor from growth after 5 doses of intravenous administration.Similar efficacy can be achieved with just a single intravenousadministration of CV890, indicating that under in vivo conditions, CV890has better efficacy than CV732 in hepatoma xenografts. In thisexperiment, 4 out of five CV890 treated mice were tumor free three weeksafter treatment. However, in CV732 group, xenografts in two mice stoppedgrowing but none of treated animals were tumor free through the six-weekexperiment. There was no tumor reduction in this group or the controlgroup of animals. By statistical analysis, the differences in meanrelative tumor volumes and serum AFP concentrations between CV890treated and CV732 treated or vehicle treated tumors are significant(p<0.01)). Taken together, these studies suggest that CV890 has asignificant antitumor activity and its oncolytic efficacy is better thanCV732, an adenovirus variant similar to AvE1a04I, in which the AFP TREwas applied to control E1A alone (Hallenback et al, 1999, Hum. Gene.Ther., 10:1721-1733).

[0403] Synergistic Antitumor Efficacy of CV890 in Combination WithChemotherapeutic Agents

[0404] In this example, different chemotherapeutic agents were tested incombination with CV890 for their in vitro killing effect in Hep3B orHepG2 cells. Drug concentrations were optimized to the extent that theywould not generate extensive cytotoxic effect on their own. Under suchconditions, some agents had shown higher cell killing effect incombination with CV890. Among them, doxorubicin, a drug currently usedin treatment of HCC showed synergistic cytotoxicity with CV890. Inexperiments using doxorubicin together with CV890 on Hep3B cells,doxorubicin at 10 ng/ml did not generate cytotoxicity, whereas CV890 atan MOI of 0.01 (pfu/cell) only had about 35% of cell killed at day 9.However, when both were applied together, 90% cells were killed 9 daysafter treatment. In order to determine the potential synergistic effectfrom the combination treatment, the MTT cell viability data weresubjected to further statistical analysis. FIG. 10 shows arepresentative IC₅₀ isobologram of doxorubicin and CV890 on Hep3B cellsat day 5. First, the dose-response curves of doxorubicin alone or CV890alone were made. Based on the original theory of Steel and Peckham(1993) and method by Aoe et al. (1999), three isoeffect curves (mode Iand mode 2a, 2b) were constructed. From this isobologram, several datapoints were in the synergy or additive area, indicating that combinationof CV890 and doxorubicin provides synergistic effect on killing of Hep3Bcells.

[0405] Although CV890 alone has good antitumor activity, we appliedcombination therapy with doxorubicin for in vivo evaluation of synergy.Animals harboring 300 mm³ Hep3B xenografts were grouped (n=6) andinjected with vehicle alone (control group), CV890 alone (1×10¹¹particles/dose, CV890 group), doxorubicin alone (10 mg/kg, doxorubicingroup), or CV890 in combination with doxorubicin (combination group).FIG. 11 shows weekly change of the relative tumor size normalized to100% at day 1. In this experiment, by week six, all animals in thecontrol group had excessive tumor which has increased by 700% ofbaseline, whereas in CV890 group and combination group, animals hadeither tumor free or tumor reduction. Of the eight Hep3B xenografts,treated with CV890, three animals (37.5%) had no palpable tumor at week5, another three animals had tumor regressed by more than 60%. Incombination group, four out of eight animals were tumor free from week5, another four animals had tumor reduction about 90%. All the animalsin the CV890 and combination group were alive and tumor was suppressedeven ten weeks following treatment whereas the control animals weresacrificed for excessive tumor burden after week 6. Furthermore, CV890also caused a drop in the serum AFP concentration in these mice.Statistical analysis shows that differences in mean relative tumorvolumes and serum AFP concentrations between CV890 and vehicle treatedgroup or combination and doxorubicin treated group are significant atdifferent times (p<0.005).

[0406] The strong efficacy in the combination treatment shows thatsingle IV injection of CV890 in combination of doxorubicin can eradicateaggressive Hep3B xenografts in most of the animals. TABLE 7 AFPproduction in different tumor cells AFP CELLS (ng/10⁶ cells/10 days)Hep3B 2645 High HepG2 3140 HuH7 4585 SNU449 60 Low PLC/PRF/5 600 Chang 0None SK-Hep1 0 HBL100 0 PA-1 0 LoVo 0

[0407] TABLE 1 IRES SEQUENCES SEQ ID NO:1 A 519 base pair IRESobtainable from encephelomycarditis virus (EMCV). 1 GAC GTCGACTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAA     SalI 51TGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGG 101GTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAG 151GAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGAC 201CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC 251AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGC 301CACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAG 351CGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGG 401GATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGG 451TTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGA            SalI 501AAAACACGAT GTCGAC GTC SEQ ID NO:2 An IRES obtainable from vascularendothelial growth factor (VEGF). 1ACGTAGTCGACAGCGCAGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCCC         SalI 51CGGCCCGGGCCTCGGTTCCAGAAGGGAGAGGAGCCCGCCAAGGCGCGCAA 101GAGAGCGGGCTGCCTCGCAGTCCGAGCCGGAGAGGGAGCGCGAGCCGCGC 151CGGCCCCGGACGGCCTCCGAAACCATGGTCGACACGTA                                   SalI SEQ ID NO:3 A 5′UTR region ofHCV. 1 GCCAGCCCCCTGATGGGGGCGACACTCCGCCATGAATCACTCCCCTGTGAGGAACTACTG 61TCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGAC 121CCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAG 181GACGACCGGGTCCTTTCTTGGATTAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCC 241GCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGG 301GTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACC (341) SEQ ID NO:4 A 5′UTRregion of BiP SEQ ID NO:4 1CCCGGGGTCACTCCTGCTGGACCTACTCCGACCCCCTAGGCCGGGAGTGAAGGCGGGACT 61TGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGC 121TGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGAAAC 181GAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGC 241CGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGC 301AGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTGGGGGAGGGTATATAAGCCGAGT 361AGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGACCTATTGGGGTGT 421TTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCTGCCCTTCGCCTGGT 481TCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGAAATTGCGCTGTGCTCC 541TGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAAGATG (595) SEQ IDNO:5 A 5′UTR of PDGF SEQ ID NO:5 1GTTTGCACCTCTCCCTGCCCGGGTGCTCGAGCTGCCGTTGCAAAGCCAACTTTGGAAAAA 61GTTTTTTGGGGGAGACTTGGGCCTTGAGGTGCCCAGCTCCGCGCTTTCCGATTTTGGGGG 121CTTTCCAGAAAATGTTGCAAAAAAGCTAAGCCGGCGGGCAGAGGAAAACGCCTGTAGCCG 181GCGAGTGAAGACGAACCATCGACTGCCGTGTTCCTTTTCCTCTTGGAGGTTGGAGTCCCC 241TGGGCGCCCCCACACCCCTAGACGCCTCGGCTGGTTCGCGACGCAGCCCCCCGGCCGTGG 301ATGCTGCACTCGGGCTCGGGATCCGCCCAGGTAGCCGGCCTCGGACCCAGGTCCTGCGCC 361CAGGTCCTCCCCTGCCCCCCAGCGACGGAGCCGGGGCCGGGGGCGGCGGCGCCGGGGGCA 421TGCGGGTGAGCCGCGGCTGCAGAGGCCTGAGCGCCTGATCGCCGCGGACCTGAGCCGAGC 481CCACCCCCCTCCCCAGCCCCCCACCCTGGCCGCGGGGGCGGCGCGCTCGATCTACGCGTC 541CGGGGCCCCGCGGGGCCGGGCCCGGAGTCGGCATG (575)

[0408] TABLE 2 LITERATURE REFERENCES FOR IRES IRES Host ExampleReference Picornavirus HAV Glass et al., 1993. Virol 193: 842-852 EMCVJang & Wimmer, 1990. Gene Dev 4: 1560-1572 Poliovirus Borman et al.,1994. EMBO J 13: 3149-3157 HCV and HCV Tsukiyama-Kohara et al., 1992. JVirol 66: 1476-1483 Leishmania LRV-1 Maga et al., 1995. Mol Cell Biol15: 4884-4889 virus Retroviruses MoMLV Torrent et al., 1996. Hum GeneTher 7: 603-612 VL30 (Harvey murine sarcoma virus) REV Lopez-Lastra etal., 1997. Hum Gene Ther 8: 1855-1865 Eukaryotic BiP Macejak & Sarnow,1991. Nature 353: 90-94 mRNA antennapedia Oh et al., 1992. Gene & Dev 6:1643-1653 mRNA FGF-2 Vagner et al., 1995. Mol Cell Biol 15: 35-44 PDGF-BBernstein et al., 1997. J Biol Chem 272: 9356-9362 IGFII Teerink et al.,1995. Biochim Biophys Acta 1264: 403-408 eIF4G Gan & Rhoads, 1996. JBiol Chem 271: 623-626 VEGF Stein et al., 1998. Mol Cell Biol 18:3112-3119; Huez et al., 1998. Mol Cell Biol 18: 6178-6190

[0409] TABLE 3 TRE SEQUENCES Nucleotide sequence of a human uroplakin II5′ flanking region. Position +1 (the translational start site) isdenoted with an asterisk. SEQ ID NO:6 (number 1 of SEQ ID NO:6corresponds to position −2239 with respect to the translational startsite). TCGATAGGTA CCCACTATAG GGCACGCGTG GTCGACGGCC CGGGCTGGTC1                                                   50 TGGCAACTTCAAGTGTGGGC CTTTCAGACC GGCATCATCA GTGTTACGGG51                                                 100 GAAGTCACTAGGAATGCAGA ATTGATTGAG CACGGTGGCT CACACCTGTA101                                                150 ATCCCAACACTCTGGGAGGC CAAGGCAGGT GGATCACTTG TGGTCAGGAG151                                                200 TTTGAGACCAGCCTGGCCAA CATGGTGAAA CCTCATCTCT ACTAAAAATA201                                                250 CAAAAATTAGCTGGGAATGG TGGCACATGC CTATAATCCC AGTTACTCAG251                                                300 GAGGCTGAGGCAGGAGAATC ATTTGAACCT GGGAGGCAGA GGTTGCAGTG301                                                350 AGCCGAGATCACGCCACTGC ACTCCAGCCT GGGTGACACA GCGAGACTCT351                                                400 GTCTCAAAAAAAAAAAAATG CAGAATTTCA GGCTTCACCC CAGACCCACT401                                                450 GCATGACTGCATGAGAAGCT GCATCTTAAC AAGATCCCTG GTAATTCATA451                                                500 CGCATATTAAATTTGGAGAT GCACTGGCGT AAGACCCTCC TACTCTCTGC501                                                550 TTAGGCCCATGAGTTCTTCC TTTACTGTCA TTCTCCACTC ACCCCAAACT551                                                600 TTGAGCCTACCCTTCCCACC TTGGCGGTAA GGACACAACC TCCCTCACAT601                                                650 TCCTACCAGGACCCTAAGCT TCCCTGGGAC TGAGGAAGAT AGAATAGTTC651                                                700 GTGGAGCAAACAGATATACA GCAACAGTCT CTGTACAGCT CTCAGGCTTC701                                                750 TGGAAGTTCTACAGCCTCTC CCGACAAAGT ATTCCACTTT CCACAAGTAA751                                                800 CTCTATGTGTCTGAGTCTCA GTTTCCACTT TTCTCTCTCT CTCTCTCTCT801                                                850 CAACTTTCTGAGACAGAGTT TCACTTAGTC GCCCAGGCTG GAGTGCAGGG851                                                900 GCACAATCTCGGCTCACTGC AACCTCCACC TCCTGGGTTC AAGTGTTTCT901                                                950 CCTGTCTCAGCCTCCCGAGT AGCTGGGATT ACAGGCACAC ACCACCGCGT951                                               1000 TAGTTTTTGTATTTTTGGTA GAGATGGTGT TTCGCCATAT TGGCCAGGCT1001                                              1050 GATCTCGAACTCCTGACCTC AGGTGATCCG CCCACCTCGG CCTCCCAAAG1051                                              1100 TGCTGGGATTACAGGCATGA GCCACCACGC CCGGCTGATC TCTTTTCTAT1101                                              1150 TTTAATAGAGATCAAACTCT CTGTGTTGCC TAGGCTGGTC TTGAACTCCT1151                                              1200 GGCCTCGAGTGATCCTCCCA CCTTGGCCTC CCAAAGTGTT GAGATTACAG1201                                              1250 GCATGAGCCACTGTGCCTGG CCTCAGTTCT ACTACAAAAG GAAGCCAGTA1251                                              1300 CCAGCTACCACCCAGGGTGG CTGTAGGGCT ACAATGGAGC ACACAGAACC1301                                              1350 CCTACCCAGGGCCCGGAAGA AGCCCCGACT CCTCTCCCCT CCCTCTGCCC1351                                              1400 AGAACTCCTCCGCTTCTTTC TGATGTAGCC CAGGGCCGGA GGAGGCAGTC1401                                              1450 AGGGAAGTTCTGTCTCTTTT TCATGTTATC TTACGAGGTC TCTTTTCTCC1451                                              1500 ATTCTCAGTCCAACAAATGG TTGCTGCCCA AGGCTGACTG TGCCCACCCC1501                                              1550 CAACCCCTGCTGGCCAGGGT CAATGTCTGT CTCTCTGGTC TCTCCAGAAG1551                                              1600 TCTTCCATGGCCACCTTCGT CCCCACCCTC CAGAGGAATC TGAAACCGCA1601                                              1650 TGTGCTCCCTGGCCCCCACA GCCCCTGCCT CTCCCAGAGC AGCAGTACCT1651                                              1700 AAGCCTCAGTGCACTCCAAG AATTGAAACC CTCAGTCTGC TGCCCCTCCC1701                                              1750 CACCAGAATGTTTCTCTCCC ATTCTTACCC ACTCAAGGCC CTTTCAGTAG1751                                              1800 CCCCTTGGAGTATTCTCTTC CTACATATCA GGGCAACTTC CAAACTCATC1801                                              1850 ACCCTTCTGAGGGGTGGGGG AAAGACCCCC ACCACATCGG GGGAGCAGTC1851                                              1900 CTCCAAGGACTGGCCAGTCT CCAGATGCCC GTGCACACAG GAACACTGCC1901                                              1950 TTATGCACGGGAGTCCCAGA AGAAGGGGTG ATTTCTTTCC CCACCTTAGT1951                                              2000 TACACCATCAAGACCCAGCC AGGGCATCCC CCCTCCTGGC CTGAGGGCCA2001                                              2050 GCTCCCCATCCTGAAAAACC TGTCTGCTCT CCCCACCCCT TTGAGGCTAT2051                                              2100 AGGGCCCAAGGGGCAGGTTG GACTGGATTC CCCTCCAGCC CCTCCCGCCC2101                                              2150 CCAGGACAAAATCAGCCACC CCAGGGGCAG GGCCTCACTT GCCTCAGGAA2151                                              2200 CCCCAGCCTGCCAGCACCTA TTCCACCTCC CAGCCCAGCA2201                                   2239 Nucleotide sequence of amouse uroplakin II 5′ flanking region. The translational start site isdenoted with an asterisk. SEQ ID NO:7 (number 1 of SEQ ID NO:7corresponds to position −3592 with respect to the translational startsite). CTCGAGGATCTCGGCCCTCTTTCTGCATCCTTGTCCTAAATCATTTTCAT1                                               50ATCTTGCTAGACCTCAGTTTGAGAGAAACGAACCTTCTCATTTTCAAGTT51                                             100GAAAAAAAAAAGAGGTTCAAAGTGGCTCACTCAAAGTTACAAGCCAACAC101                                            150TCACCACTACGAGTACAATGGCCACCATTAGTGCTGGCATGCCCCAGGAG151                                            200ACAGGCATGCATATTATTCTAGATGACTGGGAGGCAGAGGGGTGGCCTAG201                                            250TGAGGTCAGACTGTGGACAGATCAGGCAGATGTGGGTTCTGATCCCAATT251                                            300CCTCAGGCCGCAGAACTACTGTGGTTCAAGAAGGGGACAAAAGGACTGCA301                                            350GTCCGGAACAGGAGGTCCATTTGAGAGCTGACTGAGCAGAAGAGGAAAGT351                                            400GAAGAACTTCTGGGGCAAGAGCTTACCCTACTTTACAGCTTTGTTGTCTT401                                            450CTTTACTCCAGGGGCGTCCCTGGTACTCAGTAAATGTCTGTTGGCTTGAG451                                            500GAACATATGTGTAAGGAGGAAGGAGAGGGAACTTGAGGGAGTTAAGACTC501                                            550AAGAATCAATCAAGGAGAGGACAGCAGAGAAGACAGGGTTTGGGAGAGAG551                                            600ACTCCAGACATTGGCCCTGGTTCCCTTCTTGGCCACTGTGAAACCCTCCA601                                            650GAGGAACTGAGTGCTGTGGCTTTAAATGATCTCAGCACTGTCAGTGAAGC651                                            700GCTCTGCTCAAAGAGTTATCCTCTTGCTCCTGTGCCGGGGCCTCCCCCTC701                                            750CTCTCAGCTCCCAAACCCTTCTCAGCCACTGTGATGGCATAATTAGATGC751                                            800GAGAGCTCAGACCGTCAGGTCTGCTCCAGGAACCACCCATTTTCCCCAAC801                                            850CCCAGAGAAAGGTCCTAGTGGAAAAGTGGGGGCCACTGAAGGGCTGATGG851                                            900GGTTCTGTCCTTTCCCCCATGCTGGGTGGACTTAAAGTCTGCGATGTGTG900                                            950TAGGGGGTAGAAGACAACAGAACCTGGGGGCTCCGGCTGGGAGCAGGAGG951                                           1000AACTCTCACCAGACGATCTCCAAATTTACTGTGCAATGGACGATCAGGAA1001                                          1050ACTGGTTCAGATGTAGCTTCTGATACAGTGGGTCTGAGGTAAAACCCGAA1051                                          1100ACTTAATTTCTTTCAAAAATTTAAAGTTGCATTTATTATTTTATATGTGT1101                                          1150GCCCATATGTGTGCCACAGTGTCTATGTGGAGGTCAGAGGGCAAGTTGTG1151                                          1200GGCATTGGCTCTCTCCTTTCATAATGTGGCTTCTGGGGACCAAAATGTCA1201                                          1250GGCATGGTGGCAAGAGCTTTTACCTGTTGAGCCATCTCATGGTTTCGTAA1251                                          1300AACTTCCTATGACGCTTACAGGTAACGCAGAGACACAGACTCACATTTGG1301                                          1350AGTTAGCAGATGCTGTATTGGTGTAAACACTCATACACAGACACACACAC1351                                          1400ATACTCATACACACACACACACACTTATCACATGCACACACATACTCGTA1401                                          1450TACACACAGACACACACACATGCACTCTCACATTCACATATTCATACACA1451                                          1500TCCACACACACACTCATCCACACACACAGACACACATACTCATCCACACA1501                                          1550CACACACACACATACTCATACACACACACAGACACACATACTCATACACA1551                                          1600CACACAGACACACACATATAATCATACATACACAGACACACTCATACATG1601                                          1650TGCACACACACACTCATCCACACACACACACTCATACACACACACACTCA1651                                          1700TACACACACACACTCATACACACACACACGAGGTTTTTCTCAGGCTGCCT1701                                          1750TTGGGTGGAGACTGGAACTGATTTCTGTTTTTCAGCTCCTTGGCTTTTTG1751                                          1800TCCCTTTAGATGAGATCTCCTCCTCACTTTACACACAGAAAGATCACACA1801                                          1850CGAGGGAGAACTGGCGGTGCGGAAGAGGGCTACACGGTAGGGTGTCAGGG1851                                          1900TCAGGAGATCTTCCTGGCAAGTCTCAAACCTCCACATAGCACAGTGTTTA1901                                          1950CGTGAGGATTTAGGAGGAATCAGGAAGAGGATTGGTTTACTGCAGAGCAG1951                                          2000ACCATATAGGTCCACTCCTAAGCCCCATTTGAAATTAGAAGTGAGACAGT2001                                          2050GTGGGATAAAAAGAGCAGATCTCTGGTCACATTTTTAAAGGGATATGAGG2051                                          3000GTCCTGTGCCTTTAAGCCTTCCCATCTCCCTCCAATCCCCCCTCACCTTC2101                                          2150CCCACCCTAACCCTCCCCAGGTTTCTGGAGGAGCAGAGTTGCGTCTTCTC2151                                          2200CCTGCCCTGCCGAGCTGCTCACTGGCTGCTCTAGAGGCTGTGCTTTGCGG2201                                          2250TCTCCATGGAAACCATTAGTTGCTAAGCAACTGGAGCATCATCTGTGCTG2251                                          2300AGCTCAGGTCCTATCGAGTTCACCTAGCTGAGACACCCACGCCCCTGCAG2301                                          2350CCACTTTGCAGTGACAAGCCTGAGTCTCAGGTTCTGCATCTATAAAAACG2351                                          2400AGTAGCCTTTCAGGAGGGCATGCAGAGCCCCCTGGCCAGCGTCTAGAGGA2401                                          2450GAGGTGACTGAGTGGGGCCATGTCACTCGTCCATGGCTGGAGAACCTCCA2451                                          2500TCAGTCTCCCAGTTAGCCTGGGGCAGGAGAGAACCAGAGGAGCTGTGGCT2501                                          2550GCTGATTGGATGATTTACGTACCCAATCTGTTGTCCCAGGCATCGAACCC2551                                          2600CAGAGCGACCTGCACACATGCCACCGCTGCCCCGCCCTCCACCTCCTCTG2601                                          2650CTCCTGGTTACAGGATTGTTTTGTCTTGAAGGGTTTTGTTGTTGCTACTT2651                                          2700TTTGCTTTGTTTTTTCTTTTTTAACATAAGGTTTCTCTGTGTAGCCCTAG2701                                          2750CTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATC2751                                          2800CACCTTCCTCCCAAGTGCTGGGATTAAAGGCATTCGCACCATCGCCCAGC2801                                          2850CCCCGGTCTTGTTTCCTAAGGTTTTCCTGCTTTACTCGCTACCCGTTGCA2851                                          2900CAACCGCTTGCTGTCCAAGTCTGTTTGTATCTACTCCACCGCCCACTAGC2901                                          2950CTTGCTGGACTGGACCTACGTTTACCTGGAAGCCTTCACTAACTTCCCTT2951                                          3000GTCTCCACCTTCTGGAGAAATCTGAAGGCTCACACTGATACCCTCCGCTT3001                                          3050CTCCCAGAGTCGCAGTTTCTTAGGCCTCAGTTAAATACCAGAATTGGATC3051                                          3100TCAGGCTCTGCTATCCCCACCCTACCTAACCAACCCCCTCCTCTCCCATC3101                                          3150CTTACTAGCCAAAGCCCTTTCAACCCTTGGGGCTTTTCCTACACCTACAC3151                                          3200ACCAGGGCAATTTTAGAACTCATGGCTCTCCTAGAAAACGCCTACCTCCT3201                                          3250TGGAGACTGACCCTCTACAGTCCAGGAGGCAGACACTCAGACAGAGGAAC3251                                          3300TCTGTCCTTCAGTCGCGGGAGTTCCAGAAAGAGCCATACTCCCCTGCAGA3301                                          3350GCTAACTAAGCTGCCAGGACCCAGCCAGAGCATCCCCCTTTAGCCGAGGG3351                                          3400CCAGCTCCCCAGAATGAAAAACCTGTCTGGGGCCCCTCCCTGAGGCTACA3401                                          3450GTCGCCAAGGGGCAAGTTGGACTGGATTCCCAGCAGCCCCTCCCACTCCG3451                                          3500AGACAAAATCAGCTACCCTGGGGCAGGCCTCATTGGCCCCAGGAAACCCC3501                                          3550AGCCTGTCAGCACCTGTTCCAGGATCCAGTCCCAGCGCAGTA3551                                  3592 AFP-TRE. SEQ ID NO:8. 1GCATTGCTGTGAACTCTGTACTTAGGACTAAACTTTGAGCAATAACACACATAGATTGAG 61GATTGTTTGCTGTTAGCATACAAACTCTGGTTCAAAGCTCCTCTTTATTGCTTGTCTTGG 121AAAATTTGCTGTTCTTCATGGTTTCTCTTTTCACTGCTATCTATTTTTCTCAACCACTCA 181CATGGCTACAATAACTGTCTGCAAGCTTATGATTCCCAAATATCTATCTCTAGCCTCAAT 241CTTGTTCCAGAAGATAAAAAGTAGTATTCAAATGCACATCAACGTCTCCACTTGGAGGGC 301TTAAAGACGTTTCAACATACAAACCGGGGAGTTTTGCCTGGAATGTTTCCTAAAATGTGT 361CCTGTAGCACATAGGGTCCTCTTGTTCCTTAAAATCTAATTACTTTTAGCCCAGTGCTCA 421TCCCACCTATGGGGAGATGAGAGTGAAAAGGGAGCCTGATTAATAATTACACTAAGTCAA 481TAGGCATAGAGCCAGGACTGTTTGGGTAAACTGGTCACTTTATCTTAAACTAAATATATC 541CAAAACTGAACATGTACTTAGTTACTAAGTCTTTGACTTTATCTCATTCATACCACTCAG 601CTTTATCCAGGCCACTTATGAGCTCTGTGTCCTTGAACATAAAATACAAATAACCGCTAT 661GCTGTTAATTATTGGCAAATGTCCCATTTTCAACCTAAGGAAATACCATAAAGTAACAGA 721TATACCAACAAAAGGTTACTAGTTAACAGGCATTGCCTGAAAAGAGTATAAAAGAATTTC 781AGCATGATTTTCCATATTGTGCTTCCACCACTGCCAATAACA (822) Probasin-TRE SEQ IDNO:9   −426 5′-AAGCTTCCACAAGTGCATTTAGCCTCTCCAGTATTGCTGATGAATCCACAGTTCAGGTTCAATGGCGTTCAAAACTTGATCAAAAATGACCAGACTTTATATTTACACCAACATCTATCTGATTGGAGGAATGGATAATAGTCATCATGTTTAAACATCTACCATTCCAGTTAAGAAAATATGATAGCATCTTGTTCTTAGTCTTTTTCTTA                                    ARE-1ATAGGGACATAAAGCCCACAAATAAAAATATGCCTGAAGAATGGGACAGGCATTGGGCATTGTCCATGCCTAGTAAAGTACTCCAAGAACCTATTTGTATACTA                              ARE-2GATGACACAATGTCAATGTCTGTGTACAACTGCCAACTGGGATGCAAGACAC TGCCCATG CCAATCATCCTGAAAAGCAGC TATAAAAA GCAGGAAGCTACTCT        CAATbox               TATAA box     +1                        +28 GCAC CTTGTCAGTAGGTCCAGATACCTACAG-3′ Transcription site Tyrosinase-TRE. SEQ IDNO:10 PinAl end 1 CCGGT TGAAAATGATAAGTTGAATTCTGTCTTCGAGAACATAGAAAAGAA 51TTATGAAATGCCAACATGTGGTTACAAGTAATGCAGACCCAAGGCTCCCC 101AGGGACAAGAAGTCTTGTGTTAACTCTTTGTGGCTCTGAAAGAAAGAGAG 151AGAGAAAAGATTAAGCCTCCTTGTGGAGATCATGTGATGACTTCCTGATT 201CCAGCCAGAGCGAGCATTTCCATGGAAACTTCTCTTCCTCTTCACTCGAG 251ATTACTAACCTTATTGTTAATATTCTAACCATAAGAATTAAACTATTAAT 301GGTGAATAGAGTTTTTCACTTTAACATAGGCCTATCCCACTGGTGGGATA 351CGAGCCAATTCGAAAGAAAAAGTCAGTCATGTGCTTTTCAGAGGATGAAA 401GCTTAAGATAAAGACTAAAAGTGTTTGATGCTGGAGGTGGGAGTGGTATT 451ATATAGGTCTCAGCCAAGACATGTGATAATCACTGTAGTAGTAGCTGGAA 501AGAGAAATCTGTGACTCCAATTAGCCAGTTCCTGCAGACCTTGTGA                                     PinAl end Human glandularkallikrein-TRE SEQ ID NO:11 gaattcagaa ataggggaag gttgaggaag gacactgaactcaaagggga tacagtgatt 60 ggtttatttg tcttctcttc acaacattgg tgctggaggaattcccaccc tgaggttatg 120 aagatgtctg aacacccaac acatagcact ggagatatgagctcgacaag agtttctcag 180 ccacagagat tcacagccta gggcaggagg acactgtacgccaggcagaa tgacatggga 240 attgcgctca cgattggctt gaagaagcaa ggactgtgggaggtgggctt tgtagtaaca 300 agagggcagg gtgaactctg attcccatgg gggaatgtgatggtcctgtt acaaattttt 360 caagctggca gggaataaaa cccattacgg tgaggacctgtggagggcgg ctgccccaac 420 tgataaagga aatagccagg tgggggcctt tcccattgtaggggggacat atctggcaat 480 agaagccttt gagacccttt agggtacaag tactgaggcagcaaataaaa tgaaatctta 540 tttttcaact ttatactgca tgggtgtgaa gatatatttgtttctgtaca gggggtgagg 600 gaaaggaggg gaggaggaaa gttcctgcag gtctggtttggtcttgtgat ccagggggtc 660 ttggaactat ttaaattaaa ttaaattaaa acaagcgactgttttaaatt aaattaaatt 720 aaattaaatt ttactttatt ttatcttaag ttctgggctacatgtgcagg acgtgcagct 780 ttgttacata ggtaaacgtg tgccatggtg gtttgctgtacctatcaacc catcacctag 840 gtattaagcc cagcatgcat tagctgtttt tcctgacgctctccctctcc ctgactccca 900 caacaggccc cagtgtgtgt tgttcccctc cctgtgtccatgtgttctca ttgttcagct 960 cccacttata agtgagaaca tgtggtgttt ggttttctgtttctgtgtta gtttgctgag 1020 gataatggct tccacctcca tccatgttcc tgcaaaggacgtgatcttat tcttttttat 1080 ggttgcatag aaattgtttt tacaaatcca attgatattgtatttaatta caagttaatc 1140 taattagcat actagaagag attacagaag atattaggtacattgaatga ggaaatatat 1200 aaaataggac gaaggtgaaa tattaggtag gaaaagtataatagttgaaa gaagtaaaaa 1260 aaaatatgca tgagtagcag aatgtaaaag aggtgaagaacgtaatagtg actttttaga 1320 ccagattgaa ggacagagac agaaaaattt taaggaattgctaaaccatg tgagtgttag 1380 aagtacagtc aataacatta aagcctcagg aggagaaaagaataggaaag gaggaaatat 1440 gtgaataaat agtagagaca tgtttgatgg attttaaaatatttgaaaga cctcacatca 1500 aaggattcat accgtgccat tgaagaggaa gatggaaaagccaagaagcc agatgaaagt 1560 tagaaatatt attggcaaag cttaaatgtt aaaagtcctagagagaaagg atggcagaaa 1620 tattggcggg aaagaatgca gaacctagaa tataaattcatcccaacagt ttggtagtgt 1680 gcagctgtag ccttttctag ataatacact attgtcatacatcgcttaag cgagtgtaaa 1740 atggtctcct cactttattt atttatatat ttatttagttttgagatgga gcctcgctct 1800 gtctcctagg ctggagtgca atagtgcgat accactcactgcaacctctg cctcctctgt 1860 tcaagtgatt ttcttacctc agcctcccga gtagctgggattacaggtgc gtgccaccac 1920 acccggctaa tttttgtatt ttttgtagag acggggttttgccatgttgg ccaggctggt 1980 cttgaactcc tgacatcagg tgatccacct gccttggcctcctaaagtgc tgggattaca 2040 ggcatgagcc accgtgccca accactttat ttattttttatttttatttt taaatttcag 2100 cttctatttg aaatacaggg ggcacatata taggattgttacatgggtat attgaactca 2160 ggtagtgatc atactaccca acaggtaggt tttcaacccactccccctct tttcctcccc 2220 attctagtag tgtgcagtgt ctattgttct catgtttatgtctatgtgtg ctccaggttt 2280 agctcccacc tgtaagtgag aacgtgtggt atttgattttctgtccctgt gttaattcac 2340 ttaggattat ggcttccagc tccattcata ttgctgtaaaggatatgatt catttttcat 2400 ggccatgcag tattccatat tgcgtataga tcacattttctttctttttt ttttttgaga 2460 cggagtcttg ctttgctgcc taggctggag tgcagtagcacgatctcggc tcactgcaag 2520 cttcacctcc ggggttcacg tcattcttct gtctcagcttcccaagtagc tgggactaca 2580 ggcgcccgcc accacgtccg gctaattttt ttgtgtgtttttagtagaga tgggggtttc 2640 actgtgttag ccaggatggt cttgatctcc tgaccttgtggtccacctgc ctcggtctcc 2700 caaagtgctg ggattacagg ggtgagccac tgcgcccggcccatatatac cacattttct 2760 ttaaccaatc caccattgat gggcaactag gtagattccatggattccac agttttgcta 2820 ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtctttttggtat aatgatttgc 2880 attcctttgg gtatacagtc attaatagga gtgctgggttgaacggtggc tctgtttaaa 2940 attctttgag aattttccaa actgtttgcc atagagagcaaactaattta catttccacg 3000 aacagtatat aagcattccc ttttctccac agctttgtcatcatggtttt tttttttctt 3060 tattttaaaa aagaatatgt tgttgttttc ccagggtacatgtgcaggat gtgcaggttt 3120 gttacatagg tagtaaacgt gagccatggt ggtttgctgcacctgtcaac ccattacctg 3180 ggtatgaagc cctgcctgca ttagctcttt tccctaatgctctcactact gccccaccct 3240 caccctgaca gggcaaacag acaacctaca gaatgggaggaaatttttgc aatctattca 3300 tctgacaaag gtcaagaata tccagaatct acaaggaacttaagcaaatt tttacttttt 3360 aataatagcc actctgactg gcgtgaaatg gtatctcattgtggttttca tttgaatttc 3420 tctgatgatc agtgacgatg agcatttttt catatttgttggctgcttgt acgtcttttg 3480 agaagtgtct cttcatgcct tttggccact ttaatgggattattttttgc tttttagttt 3540 aagttcctta tagattctgg atattagact tcttattggatgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt ctgtttactc tattgatggcttcttttgct gtgccgaagc 3660 atcttagttt aattagaaac cacctgccaa tttttgtttttgttgcaatt gcttttgggg 3720 acttagtcat aaactctttg ccaaggtctg ggtcaagaagagtatttcct aggttttctt 3780 ctagaatttt gaaagtctga atgtaaacat ttgcatttttaatgcatctt gagttagttt 3840 ttgtatatgt gaaaggtcta ctctcatttt ctttccctctttctttcttt ctttcttttc 3900 tttctttctt tctttctttc tttctttctt tctttctttctttctttttg tccttctttc 3960 tttctttctt tctctttctt tctctctttc ttttttttttttgatggagt attgctctgt 4020 tgcccaggct gcagtgcagc ggcacgatct cggctcactgcaacctctgc ctcctgggtt 4080 caactgattc tcctgcatca gccttccaag tagctgggattataggcgcc cgccaccacg 4140 cccgactaat ttttgtattt ttagtagaga cggggttgtgccatgttggc caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctg ccttggcctcccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgt cattttctaagaggtccaag aacctcaaga 4320 tattttggga ccttgagaag agaggaattc atacaggtattacaagcaca gcctaatggc 4380 aaatctttgg catggcttgg cttcaagact ttaggctcttaaaagtcgaa tccaaaaatt 4440 tttataaaag ctccagctaa gctaccttaa aaggggcctgtatggctgat cactcttctt 4500 gctatacttt acacaaataa acaggccaaa tataatgaggccaaaattta ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggt aagaacagggaaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaattttta tatgtgcctacaatttgagc taaatcctga 4680 attattttct ggttgcaaaa actctctaaa gaagaacttggttttcattg tcttcgtgac 4740 acatttatct ggctctttac tagaacagct ttcttgtttttggtgttcta gcttgtgtgc 4800 cttacagttc tactcttcaa attattgtta tgtgtatctcatagttttcc ttcttttgag 4860 aaaactgaag ccatggtatt ctgaggacta gagatgactcaacagagctg gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgct tcaaattgctgatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcag ggaccatcatggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg ggggtagagt gtggaggtcagagcaactcc accttgaata 5100 agaaggtaaa gcaacctatc ctgaaagcta acctgccatggtggcttctg attaacctct 5160 gttctaggaa gactgacagt ttgggtctgt gtcattgcccaaatctcatg ttaaattgta 5220 atccccagtg ttcggaggtg ggacttggtg gtaggtgattcggtcatggg agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagt gagttctcgtgagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtc ctcctactgccatgtaagat acctgctcct 5400 gctttgcctt ctaccataag taaaagcccc ctgaggcctccccagaagca gatgccacca 5460 tgcttcctgt acagcctgca gaaccatcag ccaattaaacctcttttctg tataaattac 5520 cagtcttgag tatctcttta cagcagtgtg agaacggactaatacaaggg tctccaaaat 5580 tccaagttta tgtattcttt cttgccaaat agcaggtatttaccataaat cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttc aattgtcttacacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtct tgggggataatggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag acacggacat ggcttctgttggtaagtctc tattaaatgt 5820 ttctttctaa gaaactggat ttgtcagctt gtttctttggcctctcagct tcctcagact 5880 ttggggtagg ttgcacaacc ctgcccacca cgaaacaaatgtttaatatg ataaatatgg 5940 atagatataa tccacataaa taaaagctct tggagggccctcaataattg ttaagagtgt 6000 aaatgtgtcc aaagatggaa aatgtttgag aactactgtcccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagctt ggcttcttcagcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg cttcagcagt ttggggttggcttccggagc acatatttga 6180 catgttgcga ctgtgatttg gggtttggta tttgctctgaatcctaatgt ctgtccttga 6240 ggcatctaga atctgaaatc tgtggtcaga attctattatcttgagtagg acatctccag 6300 tcctggttct gccttctagg gctggagtct gtagtcagtgacccggtctg gcatttcaac 6360 ttcatataca gtgggctatc ttttggtcca tgtttcaaccaaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaat gttaaacctaggatttctgt ttaataggtt 6480 catatgaata atttcagcct gatccaactt tacattccttctaccgttat tctacaccca 6540 ccttaaaaat gcattcccaa tatattccct ggattctacctatatatggt aatcctggct 6600 ttgccagttt ctagtgcatt aacatacctg atttacattcttttacttta aagtggaaat 6660 aagagtccct ctgcagagtt caggagttct caagatggcccttacttctg acatcaattg 6720 agatttcaag ggagtcgcca agatcatcct caggttcagtgattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggc tatggtttattacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga gttgcatggg gcaaagacaaggagagctcc aagtgcagag 6900 attcctgttg ttttctccca gtggtgtcat ggaaagcagtatcttctcca tacaatgatg 6960 tgtgataata ttcagtgtat tgccaatcag ggaactcaactgagccttga ttatattgga 7020 gcttggttgc acagacatgt cgaccacctt catggctgaactttagtact tagcccctcc 7080 agacgtctac agctgatagg ctgtaaccca acattgtcaccataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaa cacaggcactctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc ccaggagctg aatgcaaagacctggcctct ttgggcaagg 7260 agaatccttt accgcacact ctccttcaca gggttattgtgaggatcaaa tgtggtcatg 7320 tgtgtgagac accagcacat gtctggctgt ggagagtgacttctatgtgt gctaacattg 7380 ctgagtgcta agaaagtatt aggcatggct ttcagcactcacagatgctc atctaatcct 7440 cacaacatgg ctacagggtg ggcactacta gcctcatttgacagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtc attctggatgcaaggggctc cagaggacca 7560 tgattagaca ttgtctgcag agaaattatg gctggatgtctctgccccgg aaagggggat 7620 gcactttcct tgacccccta tctcagatct tgactttgaggttatctcag acttcctcta 7680 tgataccagg agcccatcat aatctctctg tgtcctctccccttcctcag tcttactgcc 7740 cactcttccc agctccatct ccagctggcc aggtgtagccacagtaccta actctttgca 7800 gagaactata aatgtgtatc ctacagggga gaaaaaaaaaaagaactctg aaagagctga 7860 cattttaccg acttgcaaac acataagcta acctgccagttttgtgctgg tagaactcat 7920 gagactcctg ggtcagaggc aaaagatttt attacccacagctaaggagg cagcatgaac 7980 tttgtgttca catttgttca ctttgccccc caattcatatgggatgatca gagcagttca 8040 ggtggatgga cacaggggtt tgtggcaaag gtgagcaacctaggcttaga aatcctcaat 8100 cttataagaa ggtactagca aacttgtcca gtctttgtatctgacggaga tattatcttt 8160 ataattgggt tgaaagcaga cctactctgg aggaacatattgtatttatt gtcctgaaca 8220 gtaaacaaat ctgctgtaaa atagacgtta actttattatctaaggcagt aagcaaacct 8280 agatctgaag gcgataccat cttgcaaggc tatctgctgtacaaatatgc ttgaaaagat 8340 ggtccagaaa agaaaacggt attattgcct ttgctcagaagacacacaga aacataagag 8400 aaccatggaa aattgtctcc caacactgtt cacccagagccttccactct tgtctgcagg 8460 acagtcttaa catcccatca ttagtgtgtc taccacatctggcttcaccg tgcctaacca 8520 agatttctag gtccagttcc ccaccatgtt tggcagtgccccactgccaa ccccagaata 8580 agggagtgct cagaattccg aggggacatg ggtggggatcagaacttctg ggcttgagtg 8640 cagagggggc ccatactcct tggttccgaa ggaggaagaggctggaggtg aatgtccttg 8700 gaggggagga atgtgggttc tgaactctta aatccccaagggaggagact ggtaaggtcc 8760 cagcttccga ggtactgacg tgggaatggc ctgagaggtctaagaatccc gtatcctcgg 8820 gaaggagggg ctgaaattgt gaggggttga gttgcaggggtttgttagct tgagactcct 8880 tggtgggtcc ctgggaagca aggactggaa ccattggctccagggtttgg tgtgaaggta 8940 atgggatctc ctgattctca aagggtcaga ggactgagagttgcccatgc tttgatcttt 9000 ccatctactc cttactccac ttgagggtaa tcacctactcttctagttcc acaagagtgc 9060 gcctgcgcga gtataatctg cacatgtgcc atgtcccgaggcctggggca tcatccactc 9120 atcattcagc atctgcgcta tgcgggcgag gccggcgccatgacgtcatg tagctgcgac 9180 tatccctgca gcgcgcctct cccgtcacgt cccaaccatggagctgtgga cgtgcgtccc 9240 ctggtggatg tggcctgcgt ggtgccaggc cggggcctggtgtccgataa agatectaga 9300 accacaggaa accaggactg aaaggtgcta gagaatggccatatgtcgct gtccatgaaa 9360 tctcaaggac ttctgggtgg agggcacagg agcctgaacttacgggtttg ccccagtcca 9420 ctgtcctccc aagtgagtct cccagatacg aggcactgtgccagcatcag cttcatctgt 9480 accacatctt gtaacaggga ctacccagga ccctgatgaacaccatggtg tgtgcaggaa 9540 gagggggtga aggcatggac tcctgtgtgg tcagagcccagagggggcca tgacgggtgg 9600 ggaggaggct gtggactggc tcgagaagtg ggatgtggttgtgtttgatt tcctttggcc 9660 agataaagtg ctggatatag cattgaaaac ggagtatgaagaccagttag aatggagggt 9720 caggttggag ttgagttaca gatggggtaa aattctgcttcggatgagtt tggggattgg 9780 caatctaaag gtggtttggg atggcatggc tttgggatggaaataggttt gtttttatgt 9840 tggctgggaa gggtgtgggg attgaattgg ggatgaagtaggtttagttt tggagataga 9900 atacatggag ctggctattg catgcgagga tgtgcattagtttggtttga tctttaaata 9960 aaggaggcta ttagggttgt cttgaattag attaagttgtgttgggttga tgggttgggc 10020 ttgtgggtga tgtggttgga ttgggctgtg ttaaattggtttgggtcagg ttttggttga 10080 ggttatcatg gggatgagga tatgcttggg acatggattcaggtggttct cattcaagct 10140 gaggcaaatt tcctttcaga cggtcattcc agggaacgagtggttgtgtg ggggaaatca 10200 ggccactggc tgtgaatatc cctctatcct ggtcttgaattgtgattatc tatgtccatt 10260 ctgtctcctt cactgtactt ggaattgatc tggtcattcagctggaaatg ggggaagatt 10320 ttgtcaaatt cttgagacac agctgggtct ggatcagcgtaagccttcct tctggtttta 10380 ttgaacagat gaaatcacat tttttttttc aaaatcacagaaatcttata gagttaacag 10440 tggactctta taataagagt taacaccagg actcttattcttgattcttt tctgagacac 10500 caaaatgaga tttctcaatg ccaccctaat tctttttttttttttttttt tttttgagac 10560 acagtctggg tcttttgctc tgtcactcag gctggagcgcagtggtgtga tcatagctca 10620 ctgaaccctt gacctcctgg acttaaggga tcctcctgcttcagcctcct gagtagatgg 10680 ggctacaggt gcttgccacc acacctggct aattaaattttttttttttt tttgtagaga 10740 aagggtctca ctttgttgcc ctggctgatc ttgaacttctgacttcaagt gattcttcag 10800 ccttggactc ccaaagcact gggattgctg gcatgagccactcaccgtgc ctggcttgca 10860 gcttaatctt ggagtgtata aacctggctc ctgatagctagacatttcag tgagaaggag 10920 gcattggatt ttgcatgagg acaattctga cctaggagggcaggtcaaca ggaatccccg 10980 ctgtacctgt acgttgtaca ggcatggaga atgaggagtgaggaggccgt accggaaccc 11040 catattgttt agtggacatt ggattttgaa ataatagggaacttggtctg ggagagtcat 11100 atttctggat tggacaatat gtggtatcac aaggttttatgatgagggag aaatgtatgt 11160 ggggaaccat tttctgagtg tggaagtgca agaatcagagagtagctgaa tgccaacgct 11220 tctatttcag gaacatggta agttggaggt ccagctctcgggctcagacg ggtataggga 11280 ccaggaagtc tcacaatccg atcattctga tatttcagggcatattaggt ttggggtgca 11340 aaggaagtac ttgggactta ggcacatgag actttgtattgaaaatcaat gattggggct 11400 ggccgtggtg ctcacgcctg taatctcatc actttgggagaccgaagtgg gaggatggct 11460 tgatctcaag agttggacac cagcctaggc aacatggccagaccctctct ctacaaaaaa 11520 attaaaaatt agctggatgt ggtggtgcat gcttgtggtctcagctatcc tggaggctga 11580 gacaggagaa tcggttgagt ctgggagttc aaggctacagggagctgcga tcacgccgct 11640 gcactccagc ctgggaaaca gagtgagact gtctcagaatttttttaaaa aagaatcagt 11700 gatcatccca acccctgttg ctgttcatcc tgagcctgccttctctggct ttgttcccta 11760 gatcacatct ccatgatcca taggccctgc ccaatctgacctcacaccgt gggaatgcct 11820 ccagactgat ctagtatgtg tggaacagca agtgctggctctccctcccc ttccacagct 11880 ctgggtgtgg gagggggttg tccagcctcc agcagcatggggagggcctt ggtcagcatc 11940 taggtgccaa cagggcaagg gcggggtcct ggagaatgaaggctttatag ggctcctcag 12000 ggaggccccc cagccccaaa ctgcaccacc tggccgtggacaccggt 12047 HRE-TRE SEQ ID NO:12 ccccgagg cagtgcat gaggctcagg gcgtgcgtgagtcgcagcgagaccccg gggtgcag gccgga PSA-TRE SEQ ID NO:13 aagcttctagttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60 atctgtgacaatattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120 agagatttttgtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180 ggtgcaaccttggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240 gcctcctgagtagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300 ttttagtagagatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360 gatctgcccaccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420 ccgatatccagagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480 ttgacttttagtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540 cagcacaaatcacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600 actctaatctggcaggatac tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaaaaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaacgcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780 agggtcattgtgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840 tggggagtgccgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt gtcagggatt 900 atcttcagcacttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960 ctggcctcatttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020 gacactctggatgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080 attgtggcctgatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140 cttgaccctctcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200 ttctgtaccctcttgactct atgaccccca ccgcccactg catccagctg ggtcccctcc 1260 tatctctattcccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320 aaggggctgacattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380 tgctggcagagtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440 agcagacagcatgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500 ggtgaccagagcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560 cctgagattaggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620 tctctgatgaagatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680 catattgtatcgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740 tatctaggacagtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800 tttacaaacatccttgaaac aacaatccag aaaaaaaaag gtgttactgt ctttgctcag 1860 aagacacacagatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920 gccttccaccctttctgcag gacagtctca acgttccacc attaaatact tcttctatca 1980 catcccgcttctttatgcct aaccaaggtt ctaggtcccg atcgactgtg tctggcagca 2040 ctccactgccaaacccagaa taaggcagcg ctcaggatcc cgaaggggca tggctgggga 2100 tcagaacttctgggtttgag tgaggagtgg gtccaccctc ttgaatttca aaggaggaag 2160 aggctggatgtgaaggtact gggggaggga aagtgtcagt tccgaactct taggtcaatg 2220 agggaggagactggtaaggt cccagctccc gaggtactga tgtgggaatg gcctaagaat 2280 ctcatatcctcaggaagaag gtgctggaat cctgaggggt agagttctgg gtatatttgt 2340 ggcttaaggctctttggccc ctgaaggcag aggctggaac cattaggtcc agggtttggg 2400 gtgatagtaatgggatctct tgattcctca agagtctgag gatcgagggt tgcccattct 2460 tccatcttgccacctaatcc ttactccact tgagggtatc accagccctt ctagctccat 2520 gaaggtcccctgggcaagca caatctgagc atgaaagatg ccccagaggc cttgggtgtc 2580 atccactcatcatccagcat cacactctga gggtgtggcc agcaccatga cgtcatgttg 2640 ctgtgactatccctgcagcg tgcctctcca gccacctgcc aaccgtagag ctgcccatcc 2700 tcctctggtgggagtggcct gcatggtgcc aggctgaggc ctagtgtcag acagggagcc 2760 tggaatcatagggatccagg actcaaaagt gctagagaat ggccatatgt caccatccat 2820 gaaatctcaagggcttctgg gtggagggca cagggacctg aacttatggt ttcccaagtc 2880 tattgctctcccaagtgagt ctcccagata cgaggcactg tgccagcatc agccttatct 2940 ccaccacatcttgtaaaagg actacccagg gccctgatga acaccatggt gtgtacagga 3000 gtagggggtggaggcacgga ctcctgtgag gtcacagcca agggagcatc atcatgggtg 3060 gggaggaggcaatggacagg cttgagaacg gggatgtggt tgtatttggt tttctttggt 3120 tagataaagtgctgggtata ggattgagag tggagtatga agaccagtta ggatggagga 3180 tcagattggagttgggttag ataaagtgct gggtatagga ttgagagtgg agtatgaaga 3240 ccagttaggatggaggatca gattggagtt gggttagaga tggggtaaaa ttgtgctccg 3300 gatgagtttgggattgacac tgtggaggtg gtttgggatg gcatggcttt gggatggaaa 3360 tagatttgttttgatgttgg ctcagacatc cttggggatt gaactgggga tgaagctggg 3420 tttgattttggaggtagaag acgtggaagt agctgtcaga tttgacagtg gccatgagtt 3480 ttgtttgatggggaatcaaa caatggggga agacataagg gttggcttgt taggttaagt 3540 tgcgttgggttgatggggtc ggggctgtgt ataatgcagt tggattggtt tgtattaaat 3600 tgggttgggtcaggttttgg ttgaggatga gttgaggata tgcttgggga caccggatcc 3660 atgaggttctcactggagtg gagacaaact tcctttccag gatgaatcca gggaagcctt 3720 aattcacgtgtaggggaggt caggccactg gctaagtata tccttccact ccagctctaa 3780 gatggtcttaaattgtgatt atctatatcc acttctgtct ccctcactgt gcttggagtt 3840 tacctgatcactcaactaga aacaggggaa gattttatca aattcttttt tttttttttt 3900 tttttttgagacagagtctc actctgttgc ccaggctgga gtgcagtggc gcagtctcgg 3960 ctcactgcaacctctgcctc ccaggttcaa gtgattctcc tgcctcagcc tcctgagttg 4020 ctgggattacaggcatgcag caccatgccc agctaatttt tgtattttta gtagagatgg 4080 ggtttcaccaatgtttgcca ggctggcctc gaactcctga cctggtgatc cacctgcctc 4140 agcctcccaaagtgctggga ttacaggcgt cagccaccgc gcccagccac ttttgtcaaa 4200 ttcttgagacacagctcggg ctggatcaag tgagctactc tggttttatt gaacagctga 4260 aataaccaactttttggaaa ttgatgaaat cttacggagt taacagtgga ggtaccaggg 4320 ctcttaagagttcccgattc tcttctgaga ctacaaattg tgattttgca tgccacctta 4380 atcttttttttttttttttt aaatcgaggt ttcagtctca ttctatttcc caggctggag 4440 ttcaatagcgtgatcacagc tcactgtagc cttgaactcc tggccttaag agattctcct 4500 gcttcggtctcccaatagct aagactacag tagtccacca ccatatccag ataattttta 4560 aattttttggggggccgggc acagtggctc acgcctgtaa tcccaacacc atgggaggct 4620 gagatgggtggatcacgagg tcaggagttt gagaccagcc tgaccaacat ggtgaaactc 4680 tgtctctactaaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggc acacggcacc 4740 tgtaatcccagctactgagg aggctgaggc aggagaatca cttgaaccca gaaggcagag 4800 gttgcaatgagccgagattg cgccactgca ctccagcctg ggtgacagag tgagactctg 4860 tctcaaaaaaaaaaaatttt tttttttttt ttgtagagat ggatcttgct ttgtttctct 4920 ggttggccttgaactcctgg cttcaagtga tcctcctacc ttggcctcgg aaagtgttgg 4980 gattacaggcgtgagccacc atgactgacc tgtcgttaat cttgaggtac ataaacctgg 5040 ctcctaaaggctaaaggcta aatatttgtt ggagaagggg cattggattt tgcatgagga 5100 tgattctgacctgggagggc aggtcagcag gcatctctgt tgcacagata gagtgtacag 5160 gtctggagaacaaggagtgg ggggttattg gaattccaca ttgtttgctg cacgttggat 5220 tttgaaatgctagggaactt tgggagactc atatttctgg gctagaggat ctgtggacca 5280 caagatctttttatgatgac agtagcaatg tatctgtgga gctggattct gggttgggag 5340 tgcaaggaaaagaatgtact aaatgccaag acatctattt caggagcatg aggaataaaa 5400 gttctagtttctggtctcag agtggtgcat ggatcaggga gtctcacaat ctcctgagtg 5460 ctggtgtcttagggcacact gggtcttgga gtgcaaagga tctaggcacg tgaggctttg 5520 tatgaagaatcggggatcgt acccaccccc tgtttctgtt tcatcctggg catgtctcct 5580 ctgcctttgtcccctagatg aagtctccat gagctacaag ggcctggtgc atccagggtg 5640 atctagtaattgcagaacag caagtgctag ctctccctcc ccttccacag ctctgggtgt 5700 gggagggggttgtccagcct ccagcagcat ggggagggcc ttggtcagcc tctgggtgcc 5760 agcagggcaggggcggagtc ctggggaatg aaggttttat agggctcctg ggggaggctc 5820 cccagccccaagctt 5835 CEA TRE SEQ ID NO:14 aagcttttta gtgctttaga cagtgagctggtctgtctaa cccaagtgac ctgggctcca 60 tactcagccc cagaagtgaa gggtgaagctgggtggagcc aaaccaggca agcctaccct 120 cagggctccc agtggccctga gaaccattggacccaggacc cattacttct agggtaagga 180 aggtacaaac accagatcca accatggtctggggggacag ctgtcaaatg cctaaaaata 240 tacctgggag aggagcaggc aaactatcactgccccaggt tctctgaaca gaaacagagg 300 ggcaacccaa agtccaaatc caggtgagcaggtgcaccaa atgcccagag atatgacgag 360 gcaagaagtg aaggaaccac ccctgcatcaaatgttttgc atgggaagga gaagggggtt 420 gctcatgttc ccaatccagg agaatgcatttgggatctgc cttcttctca ctccttggtt 480 agcaagacta agcaaccagg actctggatttggggaaaga cgtttatttg tggaggccag 540 tgatgacaat cccacgaggg cctaggtgaagagggcagga aggctcgaga cactggggac 600 tgagtgaaaa ccacacccat gatctgcaccacccatggat gctccttcat tgctcacctt 660 tctgttgata tcagatggcc ccattttctgtaccttcaca gaaggacaca ggctagggtc 720 tgtgcatggc cttcatcccc ggggccatgtgaggacagca ggtgggaaag atcatgggtc 780 ctcctgggtc ctgcagggcc agaacattcatcacccatac tgacctccta gatgggaatg 840 gcttccctgg ggctgggcca acggggcctgggcaggggag aaaggacgtc aggggacagg 900 gaggaagggt catcgagacc cagcctggaaggttcttgtc tctgaccatc caggatttac 960 ttccctgcat ctacctttgg tcattttccctcagcaatga ccagctctgc ttcctgatct 1020 cagcctccca ccctggacac agcaccccagtccctggccc ggctgcatcc acccaatacc 1080 ctgataaccc aggacccatt acttctagggtaaggagggt ccaggagaca gaagctgagg 1140 aaaggtctga agaagtcaca tctgtcctggccagagggga aaaaccatca gatgctgaac 1200 caggagaatg ttgacccagg aaagggaccgaggacccaag aaaggagtca gaccaccagg 1260 gtttgcctga gaggaaggat caaggccccgagggaaagca gggctggctg catgtgcagg 1320 acactggtgg ggcatatgtg tcttagattctccctgaatt cagtgtccct gccatggcca 1380 gactctctac tcaggcctgg acatgctgaaataggacaat ggccttgtcc tctctcccca 1440 ccatttggca agagacataa aggacattccaggacatgcc ttcctgggag gtccaggttc 1500 tctgtctcac acctcaggga ctgtagttactgcatcagcc atggtaggtg ctgatctcac 1560 ccagcctgtc caggcccttc cactctccactttgtgacca tgtccaggac cacccctcaa 1620 atcctgagcc tgcaaatacc cccttgctgggtgggtggat tcagtaaaca gtgagctcct 1680 atccagccc cagagccacc tctgtcaccttcctgctggg catcatccca ccttcacaag 1740 cactaaagag catggggaga cctggctagctgggtttctg catcacaaag aaaataatcc 1800 cccaggttcg gattcccagg gctctgtatgtggagctgac agacctgagg ccaggagata 1860 gcagaggtca gccctaggga gggtgggtcatccacccagg ggacaggggt gcaccagcct 1920 tgctactgaa agggcctccc caggacagcgccatcagccc tgcctgagag ctttgctaaa 1980 cagcagtcag aggaggccat ggcagtggctgagctcctgc tccaggcccc aacagaccag 2040 accaacagca caatgcagtc cttccccaacgtcacaggtc accaaaggga aactgaggtg 2100 ctacctaacc ttagagccat caggggagataacagcccaa tttcccaaac aggccagttt 2160 caatcccatg acaatgacct ctctgctctcattcttccca aaataggacg ctgattctcc 2220 cccaccatgg atttctccct tgtcccgggagccttttctg ccccctatga tctgggcact 2280 cctgacacac acctcctctc tggtgacatatcagggtccc tcactgtcaa gcagtccaga 2340 aaggacagaa ccttggacag cgcccatctcagcttcaccc ttcctccttc acagggttca 2400 gggcaaagaa taaatggcag aggccagtgagcccagagat ggtgacaggc agtgacccag 2460 gggcagatgc ctggagcagg agctggcggggccacaggga gaaggtgatg caggaaggga 2520 aacccagaaa tgggcaggaa aggaggacacaggctctgtg gggctgcagc ccagggttgg 2580 actatgagtg tgaagccatc tcagcaagtaaggccaggtc ccatgaacaa gagtgggagc 2640 acgtggcttc ctgctctgta tatggggtgggggattccat gccccataga accagatggc 2700 cggggttcag atggagaagg agcaggacaggggatcccca ggataggagg accccagtgt 2760 ccccacccag gcaggtgact gatgaatgggcatgcagggt cctcctgggc tgggctctcc 2820 ctttgtccct caggattcct tgaaggaacatccggaagcc gaccacatct acctggtggg 2880 ttctggggag tccatgtaaa gccaggagcttgtgttgcta ggaggggtca tggcatgtgc 2940 tgggggcacc aaagagagaa acctgagggcaggcaggacc tggtctgagg aggcatggga 3000 gcccagatgg ggagatggat gtcaggaaaggctgccccat cagggagggt gatagcaatg 3060 gggggtctgt gggagtgggc acgtgggattccctgggctc tgccaagttc cctcccatag 3120 tcacaacctg gggacactgc ccatgaaggggcgcctttgc ccagccagat gctgctggtt 3180 ctgcccatcc actaccctct ctgctccagccactctgggt ctttctccag atgccctgga 3240 cagccctggc ctgggcctgt cccctgagaggtgttgggag aagctgagtc tctggggaca 3300 ctctcatcag agtctgaaag gcacatcaggaaacatccct ggtctccagg actaggcaat 3360 gaggaaaggg ccccagctcc tccctttcccactgagaggg tcgaccctgg gtggccacag 3420 tgacttctgc gtctgtccca gtcaccctgaaaccacaaca aaaccccagc cccagaccct 3480 gcaggtacaa tacatgtggg gacagtctgtacccagggga agccagttct ctcttcctag 3540 gagaccgggc ctcagggctg tgcccggggcaggcgggggc agcacgtgcc tgtccttgag 3600 aactcgggac cttaagggtc tctgctctgtgaggcacagc aaggatcctt ctgtccagag 3660 atgaaagcag ctcctgcccc tcctctgacctcttcctcct tcccaaatct caaccaacaa 3720 ataggtgttt caaatctcat catcaaatcttcatccatcc acatgagaaa gcttaaaacc 3780 caatggattg acaacatcaa gagttggaacaagtggacat ggagatgtta cttgtggaaa 3840 tttagatgtg ttcagctatc gggcaggagaatctgtgtca aattccagca tggttcagaa 3900 gaatcaaaaa gtgtcacagt ccaaatgtgcaacagtgcag gggataaaac tgtggtgcat 3960 tcaaactgag ggatattttg gaacatgagaaaggaaggga ttgctgctgc acagaacatg 4020 gatgatctca cacatagagt tgaaagaaaggagtcaatcg cagaatagaa aatgatcact 4080 aattccacct ctataaagtt tccaagaggaaaacccaatt ctgctgctag agatcagaat 4140 ggaggtgacc tgtgccttgc aatggctgtgagggtcacgg gagtgtcact tagtgcaggc 4200 aatgtgccgt atcttaatct gggcaggcctttcatgagca cataggaatg cagacattac 4260 tgctgtgttc attttacttc accggaaaagaagaataaaa tcagccgggc gcggtggctc 4320 acgcctgtaa tcccagcact ttagaagcctgaggtgggca gattacttga ggtcaggagt 4380 tcaagaccac cctggccaat atggtgaaaccccggctcta ctaaaaatac aaaaattagc 4440 tgggcatggt ggtgcgcgcc tgtaatcccagctactcggg aggctgaggc tggacaattg 4500 cttggaccca ggaagcagag gttgcagtgagccaagattg tgccactgca ctccagcttg 4560 ggcaacagag ccagactctg taaaaaaaaaaaaaaaaaaa aaaaaaagaa agaaagaaaa 4620 agaaaagaaa gtataaaatc tctttgggttaacaaaaaaa gatccacaaa acaaacacca 4680 gctcttatca aacttacaca actctgccagagaacaggaa acacaaatac tcattaactc 4740 acttttgtgg caataaaacc ttcatgtcaaaaggagacca ggacacaatg aggaagtaaa 4800 actgcaggcc ctacttgggt gcagagagggaaaatccaca aataaaacat taccagaagg 4860 agctaagatt tactgcattg agttcattccccaggtatgc aaggtgattt taacacctga 4920 aaatcaatca ttgcctttac tacatagacagattagctag aaaaaaatta caactagcag 4980 aacagaagca atttggcctt cctaaaattccacatcatat catcatgatg gagacagtgc 5040 agacgccaat gacaataaaa agagggacctccgtcacccg gtaaacatgt ccacacagct 5100 ccagcaagca cccgtcttcc cagtgaatcactgtaacctc ccctttaatc agccccaggc 5160 aaggctgcct gcgatggcca cacaggctccaacccgtggg cctcaacctc ccgcagaggc 5220 tctcctttgg ccaccccatg gggagagcatgaggacaggg cagagccctc tgatgcccac 5280 acatggcagg agctgacgcc agagccatgggggctggaga gcagagctgc tggggtcaga 5340 gcttcctgag gacacccagg cctaagggaaggcagctccc tggatggggg caaccaggct 5400 ccgggctcca acctcagagc ccgcatgggaggagccagca ctctaggcct ttcctagggt 5460 gactctgagg ggaccctgac acgacaggatcgctgaatgc acccgagatg aaggggccac 5520 cacgggaccc tgctctcgtg gcagatcaggagagagtggg acaccatgcc aggcccccat 5580 ggcatggctg cgactgaccc aggccactcccctgcatgca tcagcctcgg taagtcacat 5640 gaccaagccc aggaccaatg tggaaggaaggaaacagcat cccctttagt gatggaaccc 5700 aaggtcagtg caaagagagg ccatgagcagttaggaaggg tggtccaacc tacagcacaa 5760 accatcatct atcataagta gaagccctgctccatgaccc ctgcatttaa ataaacgttt 5820 gttaaatgag tcaaattccc tcaccatgagagctcacctg tgtgtaggcc catcacacac 5880 acaaacacac acacacacac acacacacacacacacacac acagggaaag tgcaggatcc 5940 tggacagcac caggcaggct tcacaggcagagcaaacagc gtgaatgacc catgcagtgc 6000 cctgggcccc atcagctcag agaccctgtgagggctgaga tggggctagg caggggagag 6060 acttagagag ggtggggcct ccagggagggggctgcaggg agctgggtac tgccctccag 6120 ggagggggct gcagggagct gggtactgccctccagggag ggggctgcag ggagctgggt 6180 actgccctcc agggaggggg ctgcagggagctgggtactg ccctccaggg agggggctgc 6240 agggagctgg gtactgccct ccagggaggcaggagcactg ttcccaacag agagcacatc 6300 ttcctgcagc agctgcacag acacaggagcccccatgact gccctgggcc agggtgtgga 6360 ttccaaattt cgtgccccat tgggtgggacggaggttgac cgtgacatcc aaggggcatc 6420 tgtgattcca aacttaaact actgtgcctacaaaatagga aataacccta ctttttctac 6480 tatctcaaat tccctaagca caagctagcaccctttaaat caggaagttc agtcactcct 6540 ggggtcctcc catgccccca gtctgacttgcaggtgcaca gggtggctga catctgtcct 6600 tgctcctcct cttggctcaa ctgccgcccctcctgggggt gactgatggt caggacaagg 6660 gatcctagag ctggccccat gattgacaggaaggcaggac ttggcctcca ttctgaagac 6720 taggggtgtc aagagagctg ggcatcccacagagctgcac aagatgacgc ggacagaggg 6780 tgacacaggg ctcagggctt cagacgggtcgggaggctca gctgagagtt cagggacaga 6840 cctgaggagc ctcagtggga aaagaagcactgaagtggga agttctggaa tgttctggac 6900 aagcctgagt gctctaagga aatgctcccaccccgatgta gcctgcagca ctggacggtc 6960 tgtgtacctc cccgctgccc atcctctcacagcccccgcc tctagggaca caactcctgc 7020 cctaacatgc atctttcctg tctcattccacacaaaaggg cctctggggt ccctgttctg 7080 cattgcaagg agtggaggtc acgttcccacagaccaccca gcaacagggt cctatggagg 7140 tgcggtcagg aggatcacac gtccccccatgcccagggga ctgactctgg gggtgatgga 7200 ttggcctgga ggccactggt cccctctgtccctgagggga atctgcaccc tggaggctgc 7260 cacatccctc ctgattcttt cagctgagggcccttcttga aatcccaggg aggactcaac 7320 ccccactggg aaaggcccag tgtggacggttccacagcag cccagctaag gcccttggac 7380 acagatcctg agtgagagaa cctttagggacacaggtgca cggccatgtc cccagtgccc 7440 acacagagca ggggcatctg gaccctgagtgtgtagctcc cgcgactgaa cccagccctt 7500 ccccaatgac gtgacccctg gggtggctccaggtctccag tccatgccac caaaatctcc 7560 agattgaggg tcctcccttg agtccctgatgcctgtccag gagctgcccc ctgagcaaat 7620 ctagagtgca gagggctggg attgtggcagtaaaagcagc cacatttgtc tcaggaagga 7680 aagggaggac atgagctcca ggaagggcgatggcgtcctc tagtgggcgc ctcctgttaa 7740 tgagcaaaaa ggggccagga gagttgagagatcagggctg gccttggact aaggctcaga 7800 tggagaggac tgaggtgcaa agagggggctgaagtagggg agtggtcggg agagatggga 7860 ggagcaggta aggggaagcc ccagggaggccgggggaggg tacagcagag ctctccactc 7920 ctcagcattg acatttgggg tggtcgtgctagtggggttc tgtaagttgt agggtgttca 7980 gcaccatctg gggactctac ccactaaatgccagcaggac tccctcccca agctctaaca 8040 accaacaatg tctccagact ttccaaatgtcccctggaga gcaaaattgc ttctggcaga 8100 atcactgatc tacgtcagtc tctaaaagtgactcatcagc gaaatccttc acctcttggg 8160 agaagaatca caagtgtgag aggggtagaaactgcagact tcaaaatctt tccaaaagag 8220 ttttacttaa tcagcagttt gatgtcccaggagaagatac atttagagtg tttagagttg 8280 atgccacatg gctgcctgta cctcacagcaggagcagagt gggttttcca agggcctgta 8340 accacaactg gaatgacact cactgggttacattacaaag tggaatgtgg ggaattctgt 8400 agactttggg aagggaaatg tatgacgtgagcccacagcc taaggcagtg gacagtccac 8460 tttgaggctc tcaccatcta ggagacatctcagccatgaa catagccaca tctgtcatta 8520 gaaaacatgt tttattaaga ggaaaaatctaggctagaag tgctttatgc tcttttttct 8580 ctttatgttc aaattcatat acttttagatcattccttaa agaagaatct atccccctaa 8640 gtaaatgtta tcactgactg gatagtgttggtgtctcact cccaacccct gtgtggtgac 8700 agtgccctgc ttccccagcc ctgggccctctctgattcct gagagctttg ggtgctcctt 8760 cattaggagg aagagaggaa gggtgtttttaatattctca ccattcaccc atccacctct 8820 tagacactgg gaagaatcag ttgcccactcttggatttga tcctccaatt aatgacctct 8880 atttctgtcc cttgtccatt tcaacaatgtgacaggccta agaggtgcct tctccatgtg 8940 atttttgagg agaaggttct caagataagttttctcacac ctctttgaat tacctccacc 9000 tgtgtcccca tcaccattac cagcagcatttggacccttt ttctgttagt cagatgcttt 9060 ccacctcttg agggtgtata ctgtatgctctctacacagg aatatgcaga ggaaatagaa 9120 aaagggaaat cgcattacta ttcagagagaagaagacctt tatgtgaatg aatgagagtc 9180 taaaatccta agagagccca tataaaattattaccagtgc taaaactaca aaagttacac 9240 taacagtaaa ctagaataat aaaacatgcatcacagttgc tggtaaagct aaatcagata 9300 tttttttctt agaaaaagca ttccatgtgtgttgcagtga tgacaggagt gcccttcagt 9360 caatatgctg cctgtaattt ttgttccctggcagaatgta ttgtcttttc tccctttaaa 9420 tcttaaatgc aaaactaaag gcagctcctgggccccctcc ccaaagtcag ctgcctgcaa 9480 ccagccccac gaagagcaga ggcctgagcttccctggtca aaataggggg ctagggagct 9540 taaccttgct cgataaagct gtgttcccagaatgtcgctc ctgttcccag gggcaccagc 9600 ctggagggtg gtgagcctca ctggtggcctgatgcttacc ttgtgccctc acaccagtgg 9660 tcactggaac cttgaacact tggctgtcgcccggatctgc agatgtcaag aacttctgga 9720 agtcaaatta ctgcccactt ctccagggcagatacctgtg aacatccaaa accatgccac 9780 agaaccctgc ctggggtcta caacacatatggactgtgag caccaagtcc agccctgaat 9840 ctgtgaccac ctgccaagat gcccctaactgggatccacc aatcactgca catggcaggc 9900 agcgaggctt ggaggtgctt cgccacaaggcagccccaat ttgctgggag tttcttggca 9960 cctggtagtg gtgaggagcc ttgggaccctcaggattact ccccttaagc atagtgggga 10020 cccttctgca tccccagcag gtgccccgctcttcagagcc tctctctctg aggtttaccc 10080 agacccctgc accaatgaga ccatgctgaagcctcagaga gagagatgga gctttgacca 10140 ggagccgctc ttccttgagg gccagggcagggaaagcagg aggcagcacc aggagtggga 10200 acaccagtgt ctaagcccct gatgagaacagggtggtctc tcccatatgc ccataccagg 10260 cctgtgaaca gaatcctcct tctgcagtgacaatgtctga gaggacgaca tgtttcccag 10320 cctaacgtgc agccatgccc atctacccactgcctactgc aggacagcac caacccagga 10380 gctgggaagc tgggagaaga catggaatacccatggcttc tcaccttcct ccagtccagt 10440 gggcaccatt tatgcctagg acacccacctgccggcccca ggctcttaag agttaggtca 10500 cctaggtgcc tctgggaggc cgaggcaggagaattgcttg aacccgggag gcagaggttg 10560 cagtgagccg agatcacacc actgcactccagcctgggtg acagaatgag actctgtctc 10620 aaaaaaaaag agaaagatag catcagtggctaccaagggc taggggcagg ggaaggtgga 10680 gagttaatga ttaatagtat gaagtttctatgtgagatga tgaaaatgtt ctggaaaaaa 10740 aaatatagtg gtgaggatgt agaatattgtgaatataatt aacggcattt aattgtacac 10800 ttaacatgat taatgtggca tattttatcttatgtatttg actacatcca agaaacactg 10860 ggagagggaa agcccaccat gtaaaatacacccaccctaa tcagatagtc ctcattgtac 10920 ccaggtacag gcccctcatg acctgcacaggaataactaa ggatttaagg acatgaggct 10980 tcccagccaa ctgcaggtgc acaacataaatgtatctgca aacagactga gagtaaagct 11040 gggggcacaa acctcagcac tgccaggacacacacccttc tcgtggattc tgactttatc 11100 tgacccggcc cactgtccag atcttgttgtgggattggga caagggaggt cataaagcct 11160 gtccccaggg cactctgtgt gagcacacgagacctcccca cccccccacc gttaggtctc 11220 cacacataga tctgaccatt aggcattgtgaggaggactc tagcgcgggc tcagggatca 11280 caccagagaa tcaggtacag agaggaagacggggctcgag gagctgatgg atgacacaga 11340 gcagggttcc tgcagtccac aggtccagctcaccctggtg taggtgcccc atccccctga 11400 tccaggcatc cctgacacag ctccctcccggagcctcctc ccaggtgaca catcagggtc 11460 cctcactcaa gctgtccaga gagggcagcaccttggacag cgcccacccc acttcactct 11520 tcctccctca cagggctcag ggctcagggctcaagtctca gaacaaatgg cagaggccag 11580 tgagcccaga gatggtgaca gggcaatgatccaggggcag ctgcctgaaa cgggagcagg 11640 tgaagccaca gatgggagaa gatggttcaggaagaaaaat ccaggaatgg gcaggagagg 11700 agaggaggac acaggctctg tggggctgcagcccaggatg ggactaagtg tgaagacatc 11760 tcagcaggtg aggccaggtc ccatgaacagagaagcagct cccacctccc ctgatgcacg 11820 gacacacaga gtgtgtggtg ctgtgcccccagagtcgggc tctcctgttc tggtccccag 11880 ggagtgagaa gtgaggttga cttgtccctgctcctctctg ctaccccaac attcaccttc 11940 tcctcatgcc cctctctctc aaatatgatttggatctatg tccccgccca aatctcatgt 12000 caaattgtaa accccaatgt tggaggtggggccttgtgag aagtgattgg ataatgcggg 12060 tggattttct gctttgatgc tgtttctgtgatagagatct cacatgatct ggttgtttaa 12120 aagtgtgtag cacctctccc ctctctctctctctctctta ctcatgctct gccatgtaag 12180 acgttcctgt ttccccttca ccgtccagaatgattgtaag ttttctgagg cctccccagg 12240 agcagaagcc actatgcttc ctgtacaactgcagaatgat gagcgaatta aacctctttt 12300 ctttataaat tacccagtct caggtatttctttatagcaa tgcgaggaca gactaataca 12360 atcttctact cccagatccc cgcacacgcttagccccaga catcactgcc cctgggagca 12420 tgcacagcgc agcctcctgc cgacaaaagcaaagtcacaa aaggtgacaa aaatctgcat 12480 ttggggacat ctgattgtga aagagggaggacagtacact tgtagccaca gagactgggg 12540 ctcaccgagc tgaaacctgg tagcactttggcataacatg tgcatgaccc gtgttcaatg 12600 tctagagatc agtgttgagt aaaacagcctggtctggggc cgctgctgtc cccacttccc 12660 tcctgtccac cagagggcgg cagagttcctcccaccctgg agcctcccca ggggctgctg 12720 acctccctca gccgggccca cagcccagcagggtccaccc tcacccgggt cacctcggcc 12780 cacgtcctcc tcgccctccg agctcctcacacggactctg tcagctcctc cctgcagcct 12840 atcggccgcc cacctgaggc ttgtcggccgcccacttgag gcctgtcggc tgccctctgc 12900 aggcagctcc tgtcccctac accccctccttccccgggct cagctgaaag ggcgtctccc 12960 agggcagctc cctgtgatct ccaggacagctcagtctctc acaggctccg acgcccccta 13020 tgctgtcacc tcacagccct gtcattaccattaactcctc agtcccatga agttcactga 13080 gcgcctgtct cccggttaca ggaaaactctgtgacaggga ccacgtctgt cctgctctct 13140 gtggaatccc agggcccagc ccagtgcctgacacggaaca gatgctccat aaatactggt 13200 taaatgtgtg ggagatctct aaaaagaagcatatcacctc cgtgtggccc ccagcagtca 13260 gagtctgttc catgtggaca caggggcactggcaccagca tgggaggagg ccagcaagtg 13320 cccgcggctg ccccaggaat gaggcctcaacccccagagc ttcagaaggg aggacagagg 13380 cctgcaggga atagatcctc cggcctgaccctgcagccta atccagagtt cagggtcagc 13440 tcacaccacg tcgaccctgg tcagcatccctagggcagtt ccagacaagg ccggaggtct 13500 cctcttgccc tccagggggt gacattgcacacagacatca ctcaggaaac ggattcccct 13560 ggacaggaac ctggctttgc taaggaagtggaggtggagc ctggtttcca tcccttgctc 13620 caacagaccc ttctgatctc tcccacatacctgctctgtt cctttctggg tcctatgagg 13680 accctgttct gccaggggtc cctgtgcaactccagactcc ctcctggtac caccatgggg 13740 aaggtggggt gatcacagga cagtcagcctcgcagagaca gagaccaccc aggactgtca 13800 gggagaacat ggacaggccc tgagccgcagctcagccaac agacacggag agggagggtc 13860 cccctggagc cttccccaag gacagcagagcccagagtca cccacctccc tccaccacag 13920 tcctctcttt ccaggacaca caagacacctccccctccac atgcaggatc tggggactcc 13980 tgagacctct gggcctgggt ctccatccctgggtcagtgg cggggttggt ggtactggag 14040 acagagggct ggtccctccc cagccaccacccagtgagcc tttttctagc ccccagagcc 14100 acctctgtca ccttcctgtt gggcatcatcccaccttccc agagccctgg agagcatggg 14160 gagacccggg accctgctgg gtttctctgtcacaaaggaa aataatcccc ctggtgtgac 14220 agacccaagg acagaacaca gcagaggtcagcactgggga agacaggttg tcctcccagg 14280 ggatgggggt ccatccacct tgccgaaaagatttgtctga ggaactgaaa atagaaggga 14340 aaaaagagga gggacaaaag aggcagaaatgagaggggag gggacagagg acacctgaat 14400 aaagaccaca cccatgaccc acgtgatgctgagaagtact cctgccctag gaagagactc 14460      |^(→) transcription startsite agggcagagg gaggaaggac agcagaccag acagtcacag cagccttgac aaaacgttcc14520 tggaactcaa gctcttctcc acagaggagg acagagcaga cagcagagac catggagtct14580 ccctcggccc ctccccacag atggtgcatc ccctggcaga ggctcctgct cacaggtgaa14640 gggaggacaa cctgggagag ggtgggagga gggagctggg gtctcctggg taggacaggg14700 ctgtgagacg gacagagggc tcctgttgga gcctgaatag ggaagaggac atcagagagg14760 gacaggagtc acaccagaaa aatcaaattg aactggaatt ggaaaggggc aggaaaacct14820 caagagttct attttcctag ttaattgtca ctggccacta cgtttttaaa aatcataata14880 actgcatcag atgacacttt aaataaaaac ataaccaggg catgaaacac tgtcctcatc14940 cgcctaccgc ggacattgga aaataagccc caggctgtgg agggccctag gaaccctcat15000 gaactcatcc acaggaatct gcagcctgtc ccaggcactg gggtgcaacc aagatc15056 Mucin-TRE SEQ ID NO:15 cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggctcccggtgacc actagagggc 60 gggaggagct cctggccagt ggtggagagt ggcaaggaaggaccctaggg ttcatcggag 120 cccaggttta ctcccttaag tggaaatttc ttcccccactcctccttggc tttctccaag 180 gagggaaccc aggctgctgg aaagtccggc tggggcggggactgtgggtt caggggagaa 240 cggggtgtgg aacgggacag ggagcggtta gaagggtggggctattccgg gaagtggtgg 300 ggggagggag cccaaaacta gcacctagtc cactcattatccagccctct tatttctcgg 360 ccgctctgct tcagtggacc cggggagggc ggggaagtggagtgggagac ctaggggtgg 420 gcttcccgac cttgctgtac aggacctcga cctagctggctttgttcccc atccccacgt 480 tagttgttgc cctgaggcta aaactagagc ccaggggccccaagttccag actgcccctc 540 ccccctcccc cggagccagg gagtggttgg tgaaagggggaggccagctg gagaacaaac 600 gggtagtcag ggggttgagc gattagagcc cttgtaccctacccaggaat ggttggggag 660 gaggaggaag aggtaggagg taggggaggg ggcggggttttgtcacctgt cacctgctcg 720 ctgtgcctag ggcgggcggg cggggagtgg ggggaccggtataaagcggt aggcgcctgt 780 gcccgctcca cctctcaagc agccagcgcc tgcctgaatctgttctgccc cctccccacc 840 catttcacca ccaccatg 858 αFP-TRE SEQ ID NO:16gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900gtatatatct aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260acttaggact aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800agttactaag tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160tttccaccac cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520agacccggtt ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060agatgatcag attggattac ttggccttga agatgatata tctacatcta tatctatatc 3120tatatctata tctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180aaatatgttt tgtaaagtta taaagatttc agactttata gaatctggga tttgccaaat 3240gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360catgcttggg caagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420aatacttcat gactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560atacatcgca tactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 4680attattctgc aacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 4740gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860gctgtctttt ctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220aaca 5224

[0410]

1 35 1 519 DNA Artificial Sequence IRES from encephelomycarditis virus(EMCV) 1 gacgtcgact aattccggtt attttccacc atattgccgt cttttggcaatgtgagggcc 60 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccctctcgccaaa 120 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagcttcttgaaga 180 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctggcgacaggtgc 240 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcacaaccccagtgc 300 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaagcgtattcaac 360 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatctggggcctcgg 420 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggccccccgaaccacg 480 gggacgtggt tttcctttga aaaacacgat gtcgacgtc 519 2 188 DNAArtificial Sequence IRES from vascular endothelial growth factor (VEGF)2 acgtagtcga cagcgcagag gcttggggca gccgagcggc agccaggccc cggcccgggc 60ctcggttcca gaagggagag gagcccgcca aggcgcgcaa gagagcgggc tgcctcgcag 120tccgagccgg agagggagcg cgagccgcgc cggccccgga cggcctccga aaccatggtc 180gacacgta 188 3 341 DNA Artificial Sequence 5′ UTR region of HCV 3gccagccccc tgatgggggc gacactccgc catgaatcac tcccctgtga ggaactactg 60tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180gacgaccggg tcctttcttg gattaacccg ctcaatgcct ggagatttgg gcgtgccccc 240gcaagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac c 341 4 595 DNA ArtificialSequence 5′ UTR region of BiP 4 cccggggtca ctcctgctgg acctactccgaccccctagg ccgggagtga aggcgggact 60 tgtgcggtta ccagcggaaa tgcctcggggtcagaagtcg caggagagat agacagctgc 120 tgaaccaatg ggaccagcgg atggggcggatgttatctac cattggtgaa cgttagaaac 180 gaatagcagc caatgaatca gctgggggggcggagcagtg acgtttattg cggagggggc 240 cgcttcgaat cggcggcggc cagcttggtggcctgggcca atgaacggcc tccaacgagc 300 agggccttca ccaatcggcg gcctccacgacggggctggg ggagggtata taagccgagt 360 aggcgacggt gaggtcgacg ccggccaagacagcacagac agattgacct attggggtgt 420 ttcgcgagtg tgagagggaa gcgccgcggcctgtatttct agacctgccc ttcgcctggt 480 tcgtggcgcc ttgtgacccc gggcccctgccgcctgcaag tcgaaattgc gctgtgctcc 540 tgtgctacgg cctgtggctg gactgcctgctgctgcccaa ctggctggca agatg 595 5 575 DNA Artificial Sequence 5′ UTR ofPDGF 5 gtttgcacct ctccctgccc gggtgctcga gctgccgttg caaagccaac tttggaaaaa60 gttttttggg ggagacttgg gccttgaggt gcccagctcc gcgctttccg attttggggg 120ctttccagaa aatgttgcaa aaaagctaag ccggcgggca gaggaaaacg cctgtagccg 180gcgagtgaag acgaaccatc gactgccgtg ttccttttcc tcttggaggt tggagtcccc 240tgggcgcccc cacaccccta gacgcctcgg ctggttcgcg acgcagcccc ccggccgtgg 300atgctgcact cgggctcggg atccgcccag gtagccggcc tcggacccag gtcctgcgcc 360caggtcctcc cctgcccccc agcgacggag ccggggccgg gggcggcggc gccgggggca 420tgcgggtgag ccgcggctgc agaggcctga gcgcctgatc gccgcggacc tgagccgagc 480ccacccccct ccccagcccc ccaccctggc cgcgggggcg gcgcgctcga tctacgcgtc 540cggggccccg cggggccggg cccggagtcg gcatg 575 6 2240 DNA ArtificialSequence Human uroplakin II 5′ flanking region 6 tcgataggta cccactatagggcacgcgtg gtcgacggcc cgggctggtc tggcaacttc 60 aagtgtgggc ctttcagaccggcatcatca gtgttacggg gaagtcacta ggaatgcaga 120 attgattgag cacggtggctcacacctgta atcccaacac tctgggaggc caaggcaggt 180 ggatcacttg tggtcaggagtttgagacca gcctggccaa catggtgaaa cctcatctct 240 actaaaaata caaaaattagctgggaatgg tggcacatgc ctataatccc agttactcag 300 gaggctgagg caggagaatcatttgaacct gggaggcaga ggttgcagtg agccgagatc 360 acgccactgc actccagcctgggtgacaca gcgagactct gtctcaaaaa aaaaaaaatg 420 cagaatttca ggcttcaccccagacccact gcatgactgc atgagaagct gcatcttaac 480 aagatccctg gtaattcatacgcatattaa atttggagat gcactggcgt aagaccctcc 540 tactctctgc ttaggcccatgagttcttcc tttactgtca ttctccactc accccaaact 600 ttgagcctac ccttcccaccttggcggtaa ggacacaacc tccctcacat tcctaccagg 660 accctaagct tccctgggactgaggaagat agaatagttc gtggagcaaa cagatataca 720 gcaacagtct ctgtacagctctcaggcttc tggaagttct acagcctctc ccgacaaagt 780 attccacttt ccacaagtaactctatgtgt ctgagtctca gtttccactt ttctctctct 840 ctctctctct caactttctgagacagagtt tcacttagtc gcccaggctg gagtgcaggg 900 gcacaatctc ggctcactgcaacctccacc tcctgggttc aagtgtttct cctgtctcag 960 cctcccgagt agctgggattacaggcacac accaccgcgt tagtttttgt atttttggta 1020 gagatggtgt ttcgccatattggccaggct gatctcgaac tcctgacctc aggtgatccg 1080 cccacctcgg cctcccaaagtgctgggatt acaggcatga gccaccacgc ccggctgatc 1140 tcttttctat tttaatagagatcaaactct ctgtgttgcc taggctggtc ttgaactcct 1200 ggcctcgagt gatcctcccaccttggcctc ccaaagtgtt gagattacag gcatgagcca 1260 ctgtgcctgg cctcagttctactacaaaag gaagccagta ccagctacca cccagggtgg 1320 ctgtagggct acaatggagcacacagaacc cctacccagg gcccggaaga agccccgact 1380 cctctcccct ccctctgcccagaactcctc cgcttctttc tgatgtagcc cagggccgga 1440 ggaggcagtc agggaagttctgtctctttt tcatgttatc ttacgaggtc tcttttctcc 1500 attctcagtc caacaaatggttgctgccca aggctgactg tgcccacccc caacccctgc 1560 tggccagggt caatgtctgtctctctggtc tctccagaag tcttccatgg ccaccttcgt 1620 ccccaccctc cagaggaatctgaaaccgca tgtgctccct ggcccccaca gcccctgcct 1680 ctcccagagc agcagtacctaagcctcagt gcactccaag aattgaaacc ctcagtctgc 1740 tgcccctccc caccagaatgtttctctccc attcttaccc actcaaggcc ctttcagtag 1800 ccccttggag tattctcttcctacatatca gggcaacttc caaactcatc acccttctga 1860 ggggtggggg aaagacccccaccacatcgg gggagcagtc ctccaaggac tggccagtct 1920 ccagatgccc gtgcacacaggaacactgcc ttatgcacgg gagtcccaga agaaggggtg 1980 atttctttcc ccaccttagttacaccatca agacccagcc agggcatccc ccctcctggc 2040 ctgagggcca gctccccatcctgaaaaacc tgtctgctct ccccacccct ttgaggctat 2100 agggcccaag gggcaggttggactggattc ccctccagcc cctcccgccc ccaggacaaa 2160 atcagccacc ccaggggcagggcctcactt gcctcaggaa ccccagcctg ccagcaccta 2220 ttccacctcc cagcccagca2240 7 3592 DNA Artificial Sequence Mouse uroplakin II 5′ flankingregion 7 ctcgaggatc tcggccctct ttctgcatcc ttgtcctaaa tcattttcatatcttgctag 60 acctcagttt gagagaaacg aaccttctca ttttcaagtt gaaaaaaaaaagaggttcaa 120 agtggctcac tcaaagttac aagccaacac tcaccactac gagtacaatggccaccatta 180 gtgctggcat gccccaggag acaggcatgc atattattct agatgactgggaggcagagg 240 ggtggcctag tgaggtcaga ctgtggacag atcaggcaga tgtgggttctgatcccaatt 300 cctcaggccg cagaactact gtggttcaag aaggggacaa aaggactgcagtccggaaca 360 ggaggtccat ttgagagctg actgagcaga agaggaaagt gaagaacttctggggcaaga 420 gcttacccta ctttacagct ttgttgtctt ctttactcca ggggcgtccctggtactcag 480 taaatgtctg ttggcttgag gaacatatgt gtaaggagga aggagagggaacttgaggga 540 gttaagactc aagaatcaat caaggagagg acagcagaga agacagggtttgggagagag 600 actccagaca ttggccctgg ttcccttctt ggccactgtg aaaccctccagaggaactga 660 gtgctgtggc tttaaatgat ctcagcactg tcagtgaagc gctctgctcaaagagttatc 720 ctcttgctcc tgtgccgggg cctccccctc ctctcagctc ccaaacccttctcagccact 780 gtgatggcat aattagatgc gagagctcag accgtcaggt ctgctccaggaaccacccat 840 tttccccaac cccagagaaa ggtcctagtg gaaaagtggg ggccactgaagggctgatgg 900 ggttctgtcc tttcccccat gctgggtgga cttaaagtct gcgatgtgtgtagggggtag 960 aagacaacag aacctggggg ctccggctgg gagcaggagg aactctcaccagacgatctc 1020 caaatttact gtgcaatgga cgatcaggaa actggttcag atgtagcttctgatacagtg 1080 ggtctgaggt aaaacccgaa acttaatttc tttcaaaaat ttaaagttgcatttattatt 1140 ttatatgtgt gcccatatgt gtgccacagt gtctatgtgg aggtcagagggcaagttgtg 1200 ggcattggct ctctcctttc ataatgtggc ttctggggac caaaatgtcaggcatggtgg 1260 caagagcttt tacctgttga gccatctcat ggtttcgtaa aacttcctatgacgcttaca 1320 ggtaacgcag agacacagac tcacatttgg agttagcaga tgctgtattggtgtaaacac 1380 tcatacacag acacacacac atactcatac acacacacac acacttatcacatgcacaca 1440 catactcgta tacacacaga cacacacaca tgcactctca cattcacatattcatacaca 1500 tccacacaca cactcatcca cacacacaga cacacatact catccacacacacacacaca 1560 catactcata cacacacaca gacacacata ctcatacaca cacacagacacacacatata 1620 atcatacata cacagacaca ctcatacatg tgcacacaca cactcatccacacacacaca 1680 ctcatacaca cacacactca tacacacaca cactcataca cacacacacgaggtttttct 1740 caggctgcct ttgggtggag actggaactg atttctgttt ttcagctccttggctttttg 1800 tccctttaga tgagatctcc tcctcacttt acacacagaa agatcacacacgagggagaa 1860 ctggcggtgc ggaagagggc tacacggtag ggtgtcaggg tcaggagatcttcctggcaa 1920 gtctcaaacc tccacatagc acagtgttta cgtgaggatt taggaggaatcaggaagagg 1980 attggtttac tgcagagcag accatatagg tccactccta agccccatttgaaattagaa 2040 gtgagacagt gtgggataaa aagagcagat ctctggtcac atttttaaagggatatgagg 2100 gtcctgtgcc tttaagcctt cccatctccc tccaatcccc cctcaccttccccaccctaa 2160 ccctccccag gtttctggag gagcagagtt gcgtcttctc cctgccctgccgagctgctc 2220 actggctgct ctagaggctg tgctttgcgg tctccatgga aaccattagttgctaagcaa 2280 ctggagcatc atctgtgctg agctcaggtc ctatcgagtt cacctagctgagacacccac 2340 gcccctgcag ccactttgca gtgacaagcc tgagtctcag gttctgcatctataaaaacg 2400 agtagccttt caggagggca tgcagagccc cctggccagc gtctagaggagaggtgactg 2460 agtggggcca tgtcactcgt ccatggctgg agaacctcca tcagtctcccagttagcctg 2520 gggcaggaga gaaccagagg agctgtggct gctgattgga tgatttacgtacccaatctg 2580 ttgtcccagg catcgaaccc cagagcgacc tgcacacatg ccaccgctgccccgccctcc 2640 acctcctctg ctcctggtta caggattgtt ttgtcttgaa gggttttgttgttgctactt 2700 tttgctttgt tttttctttt ttaacataag gtttctctgt gtagccctagctgtcctgga 2760 actcactctg tagaccaggc tggcctcaaa ctcagaaatc caccttcctcccaagtgctg 2820 ggattaaagg cattcgcacc atcgcccagc ccccggtctt gtttcctaaggttttcctgc 2880 tttactcgct acccgttgca caaccgcttg ctgtccaagt ctgtttgtatctactccacc 2940 gcccactagc cttgctggac tggacctacg tttacctgga agccttcactaacttccctt 3000 gtctccacct tctggagaaa tctgaaggct cacactgata ccctccgcttctcccagagt 3060 cgcagtttct taggcctcag ttaaatacca gaattggatc tcaggctctgctatccccac 3120 cctacctaac caaccccctc ctctcccatc cttactagcc aaagccctttcaacccttgg 3180 ggcttttcct acacctacac accagggcaa ttttagaact catggctctcctagaaaacg 3240 cctacctcct tggagactga ccctctacag tccaggaggc agacactcagacagaggaac 3300 tctgtccttc agtcgcggga gttccagaaa gagccatact cccctgcagagctaactaag 3360 ctgccaggac ccagccagag catccccctt tagccgaggg ccagctccccagaatgaaaa 3420 acctgtctgg ggcccctccc tgaggctaca gtcgccaagg ggcaagttggactggattcc 3480 cagcagcccc tcccactccg agacaaaatc agctaccctg gggcaggcctcattggcccc 3540 aggaaacccc agcctgtcag cacctgttcc aggatccagt cccagcgcagta 3592 8 822 DNA Artificial Sequence APF-TRE 8 gcattgctgt gaactctgtacttaggacta aactttgagc aataacacac atagattgag 60 gattgtttgc tgttagcatacaaactctgg ttcaaagctc ctctttattg cttgtcttgg 120 aaaatttgct gttcttcatggtttctcttt tcactgctat ctatttttct caaccactca 180 catggctaca ataactgtctgcaagcttat gattcccaaa tatctatctc tagcctcaat 240 cttgttccag aagataaaaagtagtattca aatgcacatc aacgtctcca cttggagggc 300 ttaaagacgt ttcaacatacaaaccgggga gttttgcctg gaatgtttcc taaaatgtgt 360 cctgtagcac atagggtcctcttgttcctt aaaatctaat tacttttagc ccagtgctca 420 tcccacctat ggggagatgagagtgaaaag ggagcctgat taataattac actaagtcaa 480 taggcataga gccaggactgtttgggtaaa ctggtcactt tatcttaaac taaatatatc 540 caaaactgaa catgtacttagttactaagt ctttgacttt atctcattca taccactcag 600 ctttatccag gccacttatgagctctgtgt ccttgaacat aaaatacaaa taaccgctat 660 gctgttaatt attggcaaatgtcccatttt caacctaagg aaataccata aagtaacaga 720 tataccaaca aaaggttactagttaacagg cattgcctga aaagagtata aaagaatttc 780 agcatgattt tccatattgtgcttccacca ctgccaataa ca 822 9 451 DNA Artificial Sequence Probasin-TRE9 aag ctt cca caa gtg cat tta gcc tct cca gta ttg ctg atg aat cca 48 cagttc agg ttc aat ggc gtt caa aac ttg atc aaa aat gac cag act 96 tta tattta cac caa cat cta tct gat tgg agg aat gga taa tag tca 144 tca tgt ttaaac atc tac cat tcc agt taa gaa aat atg ata gca tct 192 tgt tct tag tctttt tct taa tag gga cat aaa gcc cac aaa taa aaa 240 tat gcc tga aga atggga cag gca ttg ggc att gtc cat gcc tag taa 288 agt act cca aga acc tatttg tat act aga tga cac aat gtc aat gtc 336 tgt gta caa ctg cca act gggatg caa gac act gcc cat gcc aat cat 384 cct gaa aag cag cta taa aaa gcagga agc tac tct gca cct tgt cag 432 tag gtc cag ata cct aca g 451 10 546DNA Artificial Sequence Tyrosinase-TRE 10 ccggttgaaa atgataagttgaattctgtc ttcgagaaca tagaaaagaa ttatgaaatg 60 ccaacatgtg gttacaagtaatgcagaccc aaggctcccc agggacaaga agtcttgtgt 120 taactctttg tggctctgaaagaaagagag agagaaaaga ttaagcctcc ttgtggagat 180 catgtgatga cttcctgattccagccagag cgagcatttc catggaaact tctcttcctc 240 ttcactcgag attactaaccttattgttaa tattctaacc ataagaatta aactattaat 300 ggtgaataga gtttttcactttaacatagg cctatcccac tggtgggata cgagccaatt 360 cgaaagaaaa agtcagtcatgtgcttttca gaggatgaaa gcttaagata aagactaaaa 420 gtgtttgatg ctggaggtgggagtggtatt atataggtct cagccaagac atgtgataat 480 cactgtagta gtagctggaaagagaaatct gtgactccaa ttagccagtt cctgcagacc 540 ttgtga 546 11 12047 DNAArtificial Sequence Human glandular kallikrein-TRE 11 gaattcagaaataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60 ggtttatttgtcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120 aagatgtctgaacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180 ccacagagattcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240 attgcgctcacgattggctt gaagaagcaa ggactgtggg aggtgggctt tgtagtaaca 300 agagggcagggtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360 caagctggcagggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420 tgataaaggaaatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480 agaagcctttgagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540 tttttcaactttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600 gaaaggaggggaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660 ttggaactatttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720 aaattaaattttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780 ttgttacataggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840 gtattaagcccagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900 caacaggccccagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960 cccacttataagtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020 gataatggcttccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080 ggttgcatagaaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140 taattagcatactagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200 aaaataggacgaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260 aaaatatgcatgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320 ccagattgaaggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380 aagtacagtcaataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440 gtgaataaatagtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500 aaggattcataccgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560 tagaaatattattggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620 tattggcgggaaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680 gcagctgtagccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740 atggtctcctcactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800 gtctcctaggctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860 tcaagtgattttcttacctc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920 acccggctaatttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980 cttgaactcctgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040 ggcatgagccaccgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100 cttctatttgaaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160 ggtagtgatcatactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220 attctagtagtgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280 agctcccacctgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340 ttaggattatggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400 ggccatgcagtattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460 cggagtcttgctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520 cttcacctccggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580 ggcgcccgccaccacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640 actgtgttagccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700 caaagtgctgggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760 ttaaccaatccaccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820 ttgtgtgcagtgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880 attcctttgggtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940 attctttgagaattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000 aacagtatataagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060 tattttaaaaaagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120 gttacataggtagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180 ggtatgaagccctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240 caccctgacagggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300 tctgacaaaggtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360 aataatagccactctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420 tctgatgatcagtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg 3480 agaagtgtctcttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540 aagttccttatagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600 tcttccattctgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660 atcttagtttaattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720 acttagtcataaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780 ctagaattttgaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840 ttgtatatgtgaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900 tttctttctttctttctttc tttctttctt tctttctttc tttctttttg tccttctttc 3960 tttctttctttctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020 tgcccaggctgcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080 caactgattctcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140 cccgactaatttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200 tgaaactcctgacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260 gtgtgagccactgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320 tattttgggaccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380 aaatctttggcatggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440 tttataaaagctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500 gctatactttacacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560 aaattggtcctgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620 ttgcatctgacctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680 attattttctggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740 acatttatctggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800 cttacagttctactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860 aaaactgaagccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920 tcatatgcaatccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980 atacatttaaaaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagagccagattcgg ggggtagagt gtggaggtca gagcaactcc accttgaata 5100 agaaggtaaagcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160 gttctaggaagactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220 atccccagtgttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280 cttctttgtggtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340 tgtgtggcccctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400 gctttgccttctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgtacagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520 cagtcttgagtatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580 tccaagtttatgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640 ggtcaaacaaccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700 cctcccctatggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760 ttgtctggctgccacctgag acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaagaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880 ttggggtaggttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940 atagatataatccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000 aaatgtgtccaaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060 tagagtgtgggaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtctgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180 catgttgcgactgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240 ggcatctagaatctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300 tcctggttctgccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360 ttcatatacagtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420 agaacctttccccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480 catatgaataatttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540 ccttaaaaatgcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600 ttgccagtttctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660 aagagtccctctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720 agatttcaagggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780 taactcaatgaaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840 gaaaatctagcaagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttgttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960 tgtgataatattcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020 gcttggttgcacagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080 agacgtctacagctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140 tatccagtgtggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200 aaaagcttagagatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260 agaatcctttaccgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320 tgtgtgagacaccagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380 ctgagtgctaagaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440 cacaacatggctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500 taagaagggggtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560 tgattagacattgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620 gcactttccttgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680 tgataccaggagcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740 cactcttcccagctccatct ccagctggcc aggtgtagcc acagtaccta actctttgca 7800 gagaactataaatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860 cattttaccgacttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920 gagactcctgggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980 tttgtgttcacatttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040 ggtggatggacacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100 cttataagaaggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160 ataattgggttgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220 gtaaacaaatctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280 agatctgaaggcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340 ggtccagaaaagaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400 aaccatggaaaattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460 acagtcttaacatcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520 agatttctaggtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580 agggagtgctcagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640 cagagggggcccatactcct tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700 gaggggaggaatgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760 cagcttccgaggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820 gaaggaggggctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880 tggtgggtccctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940 atgggatctcctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000 ccatctactccttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060 gcctgcgcgagtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120 atcattcagcatctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180 tatccctgcagcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240 ctggtggatgtggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300 accacaggaaaccaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360 tctcaaggacttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420 ctgtcctcccaagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480 accacatcttgtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540 gagggggtgaaggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600 ggaggaggctgtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660 agataaagtgctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720 caggttggagttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780 caatctaaaggtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840 tggctgggaagggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900 atacatggagctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960 aaggaggctattagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020 ttgtgggtgatgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080 ggttatcatggggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140 gaggcaaatttcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200 ggccactggctgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260 ctgtctccttcactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320 ttgtcaaattcttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380 ttgaacagatgaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440 tggactcttataataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500 caaaatgagatttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560 acagtctgggtcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620 ctgaacccttgacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680 ggctacaggtgcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740 aagggtctcactttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800 ccttggactcccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860 gcttaatcttggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920 gcattggattttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980 ctgtacctgtacgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040 catattgtttagtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100 atttctggattggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160 ggggaaccattttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220 tctatttcaggaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280 ccaggaagtctcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340 aaggaagtacttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct 11400 ggccgtggtgctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460 tgatctcaagagttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520 attaaaaattagctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580 gacaggagaatcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640 gcactccagcctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 11700 gatcatcccaacccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760 gatcacatctccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820 ccagactgatctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880 ctgggtgtgggagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940 taggtgccaacagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000 ggaggccccccagccccaaa ctgcaccacc tggccgtgga caccggt 12047 12 67 DNA ArtificialSequence HRE-TRE 12 ccccgaggca gtgcatgagg ctcagggcgt gcgtgagtcgcagcgagacc ccggggtgca 60 ggccgga 67 13 5835 DNA Artificial SequencePSA-TRE 13 aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatctctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagccttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggctggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattctcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaatttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctcctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagccaccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgactgtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggctcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggtgaacaaaggg 600 actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaagaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacctctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacagtctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcacatgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgtgtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatgggatgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggaccactagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgcagagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcactaacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactaggaccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctgggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtcagtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccagagttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactgctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gccccccccaaatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccactggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgctcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctactctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagacattatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaaggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgtctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctgttcagccaga 1920 gccttccacc ctttctgcag gacagtctca acgttccacc attaaatacttcttctatca 1980 catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtgtctggcagca 2040 ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggcatggctgggga 2100 tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttcaaaggaggaag 2160 aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactcttaggtcaatg 2220 agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatggcctaagaat 2280 ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgggtatatttgt 2340 ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtccagggtttggg 2400 gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggttgcccattct 2460 tccatcttgc cacctaatcc ttactccact tgagggtatc accagcccttctagctccat 2520 gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggccttgggtgtc 2580 atccactcat catccagcat cacactctga gggtgtggcc agcaccatgacgtcatgttg 2640 ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagagctgcccatcc 2700 tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcagacagggagcc 2760 tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgtcaccatccat 2820 gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggtttcccaagtc 2880 tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatcagccttatct 2940 ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggtgtgtacagga 3000 gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatcatcatgggtg 3060 gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggttttctttggt 3120 tagataaagt gctgggtata ggattgagag tggagtatga agaccagttaggatggagga 3180 tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtggagtatgaaga 3240 ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaattgtgctccg 3300 gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggctttgggatggaaa 3360 tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactggggatgaagctggg 3420 tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtggccatgagtt 3480 ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgttaggttaagt 3540 tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtttgtattaaat 3600 tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttggggacaccggatcc 3660 atgaggttct cactggagtg gagacaaact tcctttccag gatgaatccagggaagcctt 3720 aattcacgtg taggggaggt caggccactg gctaagtata tccttccactccagctctaa 3780 gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgtgcttggagtt 3840 tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttttttttttttt 3900 tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggcgcagtctcgg 3960 ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcctcctgagttg 4020 ctgggattac aggcatgcag caccatgccc agctaatttt tgtatttttagtagagatgg 4080 ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatccacctgcctc 4140 agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccacttttgtcaaa 4200 ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttattgaacagctga 4260 aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtggaggtaccaggg 4320 ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgcatgccacctta 4380 atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcccaggctggag 4440 ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaagagattctcct 4500 gcttcggtct cccaatagct aagactacag tagtccacca ccatatccagataattttta 4560 aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacaccatgggaggct 4620 gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacatggtgaaactc 4680 tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggcacacggcacc 4740 tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaacccagaaggcagag 4800 gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagagtgagactctg 4860 tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgctttgtttctct 4920 ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcggaaagtgttgg 4980 gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtacataaacctgg 5040 ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattttgcatgagga 5100 tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagatagagtgtacag 5160 gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctgcacgttggat 5220 tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggatctgtggacca 5280 caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattctgggttgggag 5340 tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatgaggaataaaa 5400 gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaatctcctgagtg 5460 ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacgtgaggctttg 5520 tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctgggcatgtctcct 5580 ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgcatccagggtg 5640 atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacagctctgggtgt 5700 gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcctctgggtgcc 5760 agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctgggggaggctc 5820 cccagcccca agctt 5835 14 15056 DNA Artificial SequenceCEA TRE 14 aagcttttta gtgctttaga cagtgagctg gtctgtctaa cccaagtgacctgggctcca 60 tactcagccc cagaagtgaa gggtgaagct gggtggagcc aaaccaggcaagcctaccct 120 cagggctccc agtggcctga gaaccattgg acccaggacc cattacttctagggtaagga 180 aggtacaaac accagatcca accatggtct ggggggacag ctgtcaaatgcctaaaaata 240 tacctgggag aggagcaggc aaactatcac tgccccaggt tctctgaacagaaacagagg 300 ggcaacccaa agtccaaatc caggtgagca ggtgcaccaa atgcccagagatatgacgag 360 gcaagaagtg aaggaaccac ccctgcatca aatgttttgc atgggaaggagaagggggtt 420 gctcatgttc ccaatccagg agaatgcatt tgggatctgc cttcttctcactccttggtt 480 agcaagacta agcaaccagg actctggatt tggggaaaga cgtttatttgtggaggccag 540 tgatgacaat cccacgaggg cctaggtgaa gagggcagga aggctcgagacactggggac 600 tgagtgaaaa ccacacccat gatctgcacc acccatggat gctccttcattgctcacctt 660 tctgttgata tcagatggcc ccattttctg taccttcaca gaaggacacaggctagggtc 720 tgtgcatggc cttcatcccc ggggccatgt gaggacagca ggtgggaaagatcatgggtc 780 ctcctgggtc ctgcagggcc agaacattca tcacccatac tgacctcctagatgggaatg 840 gcttccctgg ggctgggcca acggggcctg ggcaggggag aaaggacgtcaggggacagg 900 gaggaagggt catcgagacc cagcctggaa ggttcttgtc tctgaccatccaggatttac 960 ttccctgcat ctacctttgg tcattttccc tcagcaatga ccagctctgcttcctgatct 1020 cagcctccca ccctggacac agcaccccag tccctggccc ggctgcatccacccaatacc 1080 ctgataaccc aggacccatt acttctaggg taaggagggt ccaggagacagaagctgagg 1140 aaaggtctga agaagtcaca tctgtcctgg ccagagggga aaaaccatcagatgctgaac 1200 caggagaatg ttgacccagg aaagggaccg aggacccaag aaaggagtcagaccaccagg 1260 gtttgcctga gaggaaggat caaggccccg agggaaagca gggctggctgcatgtgcagg 1320 acactggtgg ggcatatgtg tcttagattc tccctgaatt cagtgtccctgccatggcca 1380 gactctctac tcaggcctgg acatgctgaa ataggacaat ggccttgtcctctctcccca 1440 ccatttggca agagacataa aggacattcc aggacatgcc ttcctgggaggtccaggttc 1500 tctgtctcac acctcaggga ctgtagttac tgcatcagcc atggtaggtgctgatctcac 1560 ccagcctgtc caggcccttc cactctccac tttgtgacca tgtccaggaccacccctcag 1620 atcctgagcc tgcaaatacc cccttgctgg gtgggtggat tcagtaaacagtgagctcct 1680 atccagcccc cagagccacc tctgtcacct tcctgctggg catcatcccaccttcacaag 1740 cactaaagag catggggaga cctggctagc tgggtttctg catcacaaagaaaataatcc 1800 cccaggttcg gattcccagg gctctgtatg tggagctgac agacctgaggccaggagata 1860 gcagaggtca gccctaggga gggtgggtca tccacccagg ggacaggggtgcaccagcct 1920 tgctactgaa agggcctccc caggacagcg ccatcagccc tgcctgagagctttgctaaa 1980 cagcagtcag aggaggccat ggcagtggct gagctcctgc tccaggccccaacagaccag 2040 accaacagca caatgcagtc cttccccaac gtcacaggtc accaaagggaaactgaggtg 2100 ctacctaacc ttagagccat caggggagat aacagcccaa tttcccaaacaggccagttt 2160 caatcccatg acaatgacct ctctgctctc attcttccca aaataggacgctgattctcc 2220 cccaccatgg atttctccct tgtcccggga gccttttctg ccccctatgatctgggcact 2280 cctgacacac acctcctctc tggtgacata tcagggtccc tcactgtcaagcagtccaga 2340 aaggacagaa ccttggacag cgcccatctc agcttcaccc ttcctccttcacagggttca 2400 gggcaaagaa taaatggcag aggccagtga gcccagagat ggtgacaggcagtgacccag 2460 gggcagatgc ctggagcagg agctggcggg gccacaggga gaaggtgatgcaggaaggga 2520 aacccagaaa tgggcaggaa aggaggacac aggctctgtg gggctgcagcccagggttgg 2580 actatgagtg tgaagccatc tcagcaagta aggccaggtc ccatgaacaagagtgggagc 2640 acgtggcttc ctgctctgta tatggggtgg gggattccat gccccatagaaccagatggc 2700 cggggttcag atggagaagg agcaggacag gggatcccca ggataggaggaccccagtgt 2760 ccccacccag gcaggtgact gatgaatggg catgcagggt cctcctgggctgggctctcc 2820 ctttgtccct caggattcct tgaaggaaca tccggaagcc gaccacatctacctggtggg 2880 ttctggggag tccatgtaaa gccaggagct tgtgttgcta ggaggggtcatggcatgtgc 2940 tgggggcacc aaagagagaa acctgagggc aggcaggacc tggtctgaggaggcatggga 3000 gcccagatgg ggagatggat gtcaggaaag gctgccccat cagggagggtgatagcaatg 3060 gggggtctgt gggagtgggc acgtgggatt ccctgggctc tgccaagttccctcccatag 3120 tcacaacctg gggacactgc ccatgaaggg gcgcctttgc ccagccagatgctgctggtt 3180 ctgcccatcc actaccctct ctgctccagc cactctgggt ctttctccagatgccctgga 3240 cagccctggc ctgggcctgt cccctgagag gtgttgggag aagctgagtctctggggaca 3300 ctctcatcag agtctgaaag gcacatcagg aaacatccct ggtctccaggactaggcaat 3360 gaggaaaggg ccccagctcc tccctttgcc actgagaggg tcgaccctgggtggccacag 3420 tgacttctgc gtctgtccca gtcaccctga aaccacaaca aaaccccagccccagaccct 3480 gcaggtacaa tacatgtggg gacagtctgt acccagggga agccagttctctcttcctag 3540 gagaccgggc ctcagggctg tgcccggggc aggcgggggc agcacgtgcctgtccttgag 3600 aactcgggac cttaagggtc tctgctctgt gaggcacagc aaggatccttctgtccagag 3660 atgaaagcag ctcctgcccc tcctctgacc tcttcctcct tcccaaatctcaaccaacaa 3720 ataggtgttt caaatctcat catcaaatct tcatccatcc acatgagaaagcttaaaacc 3780 caatggattg acaacatcaa gagttggaac aagtggacat ggagatgttacttgtggaaa 3840 tttagatgtg ttcagctatc gggcaggaga atctgtgtca aattccagcatggttcagaa 3900 gaatcaaaaa gtgtcacagt ccaaatgtgc aacagtgcag gggataaaactgtggtgcat 3960 tcaaactgag ggatattttg gaacatgaga aaggaaggga ttgctgctgcacagaacatg 4020 gatgatctca cacatagagt tgaaagaaag gagtcaatcg cagaatagaaaatgatcact 4080 aattccacct ctataaagtt tccaagagga aaacccaatt ctgctgctagagatcagaat 4140 ggaggtgacc tgtgccttgc aatggctgtg agggtcacgg gagtgtcacttagtgcaggc 4200 aatgtgccgt atcttaatct gggcagggct ttcatgagca cataggaatgcagacattac 4260 tgctgtgttc attttacttc accggaaaag aagaataaaa tcagccgggcgcggtggctc 4320 acgcctgtaa tcccagcact ttagaaggct gaggtgggca gattacttgaggtcaggagt 4380 tcaagaccac cctggccaat atggtgaaac cccggctcta ctaaaaatacaaaaattagc 4440 tgggcatggt ggtgcgcgcc tgtaatccca gctactcggg aggctgaggctggacaattg 4500 cttggaccca ggaagcagag gttgcagtga gccaagattg tgccactgcactccagcttg 4560 ggcaacagag ccagactctg taaaaaaaaa aaaaaaaaaa aaaaaaagaaagaaagaaaa 4620 agaaaagaaa gtataaaatc tctttgggtt aacaaaaaaa gatccacaaaacaaacacca 4680 gctcttatca aacttacaca actctgccag agaacaggaa acacaaatactcattaactc 4740 acttttgtgg caataaaacc ttcatgtcaa aaggagacca ggacacaatgaggaagtaaa 4800 actgcaggcc ctacttgggt gcagagaggg aaaatccaca aataaaacattaccagaagg 4860 agctaagatt tactgcattg agttcattcc ccaggtatgc aaggtgattttaacacctga 4920 aaatcaatca ttgcctttac tacatagaca gattagctag aaaaaaattacaactagcag 4980 aacagaagca atttggcctt cctaaaattc cacatcatat catcatgatggagacagtgc 5040 agacgccaat gacaataaaa agagggacct ccgtcacccg gtaaacatgtccacacagct 5100 ccagcaagca cccgtcttcc cagtgaatca ctgtaacctc ccctttaatcagccccaggc 5160 aaggctgcct gcgatggcca cacaggctcc aacccgtggg cctcaacctcccgcagaggc 5220 tctcctttgg ccaccccatg gggagagcat gaggacaggg cagagccctctgatgcccac 5280 acatggcagg agctgacgcc agagccatgg gggctggaga gcagagctgctggggtcaga 5340 gcttcctgag gacacccagg cctaagggaa ggcagctccc tggatgggggcaaccaggct 5400 ccgggctcca acctcagagc ccgcatggga ggagccagca ctctaggcctttcctagggt 5460 gactctgagg ggaccctgac acgacaggat cgctgaatgc acccgagatgaaggggccac 5520 cacgggaccc tgctctcgtg gcagatcagg agagagtggg acaccatgccaggcccccat 5580 ggcatggctg cgactgaccc aggccactcc cctgcatgca tcagcctcggtaagtcacat 5640 gaccaagccc aggaccaatg tggaaggaag gaaacagcat cccctttagtgatggaaccc 5700 aaggtcagtg caaagagagg ccatgagcag ttaggaaggg tggtccaacctacagcacaa 5760 accatcatct atcataagta gaagccctgc tccatgaccc ctgcatttaaataaacgttt 5820 gttaaatgag tcaaattccc tcaccatgag agctcacctg tgtgtaggcccatcacacac 5880 acaaacacac acacacacac acacacacac acacacacac acagggaaagtgcaggatcc 5940 tggacagcac caggcaggct tcacaggcag agcaaacagc gtgaatgacccatgcagtgc 6000 cctgggcccc atcagctcag agaccctgtg agggctgaga tggggctaggcaggggagag 6060 acttagagag ggtggggcct ccagggaggg ggctgcaggg agctgggtactgccctccag 6120 ggagggggct gcagggagct gggtactgcc ctccagggag ggggctgcagggagctgggt 6180 actgccctcc agggaggggg ctgcagggag ctgggtactg ccctccagggagggggctgc 6240 agggagctgg gtactgccct ccagggaggc aggagcactg ttcccaacagagagcacatc 6300 ttcctgcagc agctgcacag acacaggagc ccccatgact gccctgggccagggtgtgga 6360 ttccaaattt cgtgccccat tgggtgggac ggaggttgac cgtgacatccaaggggcatc 6420 tgtgattcca aacttaaact actgtgccta caaaatagga aataaccctactttttctac 6480 tatctcaaat tccctaagca caagctagca ccctttaaat caggaagttcagtcactcct 6540 ggggtcctcc catgccccca gtctgacttg caggtgcaca gggtggctgacatctgtcct 6600 tgctcctcct cttggctcaa ctgccgcccc tcctgggggt gactgatggtcaggacaagg 6660 gatcctagag ctggccccat gattgacagg aaggcaggac ttggcctccattctgaagac 6720 taggggtgtc aagagagctg ggcatcccac agagctgcac aagatgacgcggacagaggg 6780 tgacacaggg ctcagggctt cagacgggtc gggaggctca gctgagagttcagggacaga 6840 cctgaggagc ctcagtggga aaagaagcac tgaagtggga agttctggaatgttctggac 6900 aagcctgagt gctctaagga aatgctccca ccccgatgta gcctgcagcactggacggtc 6960 tgtgtacctc cccgctgccc atcctctcac agcccccgcc tctagggacacaactcctgc 7020 cctaacatgc atctttcctg tctcattcca cacaaaaggg cctctggggtccctgttctg 7080 cattgcaagg agtggaggtc acgttcccac agaccaccca gcaacagggtcctatggagg 7140 tgcggtcagg aggatcacac gtccccccat gcccagggga ctgactctgggggtgatgga 7200 ttggcctgga ggccactggt cccctctgtc cctgagggga atctgcaccctggaggctgc 7260 cacatccctc ctgattcttt cagctgaggg cccttcttga aatcccagggaggactcaac 7320 ccccactggg aaaggcccag tgtggacggt tccacagcag cccagctaaggcccttggac 7380 acagatcctg agtgagagaa cctttaggga cacaggtgca cggccatgtccccagtgccc 7440 acacagagca ggggcatctg gaccctgagt gtgtagctcc cgcgactgaacccagccctt 7500 ccccaatgac gtgacccctg gggtggctcc aggtctccag tccatgccaccaaaatctcc 7560 agattgaggg tcctcccttg agtccctgat gcctgtccag gagctgccccctgagcaaat 7620 ctagagtgca gagggctggg attgtggcag taaaagcagc cacatttgtctcaggaagga 7680 aagggaggac atgagctcca ggaagggcga tggcgtcctc tagtgggcgcctcctgttaa 7740 tgagcaaaaa ggggccagga gagttgagag atcagggctg gccttggactaaggctcaga 7800 tggagaggac tgaggtgcaa agagggggct gaagtagggg agtggtcgggagagatggga 7860 ggagcaggta aggggaagcc ccagggaggc cgggggaggg tacagcagagctctccactc 7920 ctcagcattg acatttgggg tggtcgtgct agtggggttc tgtaagttgtagggtgttca 7980 gcaccatctg gggactctac ccactaaatg ccagcaggac tccctccccaagctctaaca 8040 accaacaatg tctccagact ttccaaatgt cccctggaga gcaaaattgcttctggcaga 8100 atcactgatc tacgtcagtc tctaaaagtg actcatcagc gaaatccttcacctcttggg 8160 agaagaatca caagtgtgag aggggtagaa actgcagact tcaaaatctttccaaaagag 8220 ttttacttaa tcagcagttt gatgtcccag gagaagatac atttagagtgtttagagttg 8280 atgccacatg gctgcctgta cctcacagca ggagcagagt gggttttccaagggcctgta 8340 accacaactg gaatgacact cactgggtta cattacaaag tggaatgtggggaattctgt 8400 agactttggg aagggaaatg tatgacgtga gcccacagcc taaggcagtggacagtccac 8460 tttgaggctc tcaccatcta ggagacatct cagccatgaa catagccacatctgtcatta 8520 gaaaacatgt tttattaaga ggaaaaatct aggctagaag tgctttatgctcttttttct 8580 ctttatgttc aaattcatat acttttagat cattccttaa agaagaatctatccccctaa 8640 gtaaatgtta tcactgactg gatagtgttg gtgtctcact cccaacccctgtgtggtgac 8700 agtgccctgc ttccccagcc ctgggccctc tctgattcct gagagctttgggtgctcctt 8760 cattaggagg aagagaggaa gggtgttttt aatattctca ccattcacccatccacctct 8820 tagacactgg gaagaatcag ttgcccactc ttggatttga tcctcgaattaatgacctct 8880 atttctgtcc cttgtccatt tcaacaatgt gacaggccta agaggtgccttctccatgtg 8940 atttttgagg agaaggttct caagataagt tttctcacac ctctttgaattacctccacc 9000 tgtgtcccca tcaccattac cagcagcatt tggacccttt ttctgttagtcagatgcttt 9060 ccacctcttg agggtgtata ctgtatgctc tctacacagg aatatgcagaggaaatagaa 9120 aaagggaaat cgcattacta ttcagagaga agaagacctt tatgtgaatgaatgagagtc 9180 taaaatccta agagagccca tataaaatta ttaccagtgc taaaactacaaaagttacac 9240 taacagtaaa ctagaataat aaaacatgca tcacagttgc tggtaaagctaaatcagata 9300 tttttttctt agaaaaagca ttccatgtgt gttgcagtga tgacaggagtgcccttcagt 9360 caatatgctg cctgtaattt ttgttccctg gcagaatgta ttgtcttttctccctttaaa 9420 tcttaaatgc aaaactaaag gcagctcctg ggccccctcc ccaaagtcagctgcctgcaa 9480 ccagccccac gaagagcaga ggcctgagct tccctggtca aaatagggggctagggagct 9540 taaccttgct cgataaagct gtgttcccag aatgtcgctc ctgttcccaggggcaccagc 9600 ctggagggtg gtgagcctca ctggtggcct gatgcttacc ttgtgccctcacaccagtgg 9660 tcactggaac cttgaacact tggctgtcgc ccggatctgc agatgtcaagaacttctgga 9720 agtcaaatta ctgcccactt ctccagggca gatacctgtg aacatccaaaaccatgccac 9780 agaaccctgc ctggggtcta caacacatat ggactgtgag caccaagtccagccctgaat 9840 ctgtgaccac ctgccaagat gcccctaact gggatccacc aatcactgcacatggcaggc 9900 agcgaggctt ggaggtgctt cgccacaagg cagccccaat ttgctgggagtttcttggca 9960 cctggtagtg gtgaggagcc ttgggaccct caggattact ccccttaagcatagtgggga 10020 cccttctgca tccccagcag gtgccccgct cttcagagcc tctctctctgaggtttaccc 10080 agacccctgc accaatgaga ccatgctgaa gcctcagaga gagagatggagctttgacca 10140 ggagccgctc ttccttgagg gccagggcag ggaaagcagg aggcagcaccaggagtggga 10200 acaccagtgt ctaagcccct gatgagaaca gggtggtctc tcccatatgcccataccagg 10260 cctgtgaaca gaatcctcct tctgcagtga caatgtctga gaggacgacatgtttcccag 10320 cctaacgtgc agccatgccc atctacccac tgcctactgc aggacagcaccaacccagga 10380 gctgggaagc tgggagaaga catggaatac ccatggcttc tcaccttcctccagtccagt 10440 gggcaccatt tatgcctagg acacccacct gccggcccca ggctcttaagagttaggtca 10500 cctaggtgcc tctgggaggc cgaggcagga gaattgcttg aacccgggaggcagaggttg 10560 cagtgagccg agatcacacc actgcactcc agcctgggtg acagaatgagactctgtctc 10620 aaaaaaaaag agaaagatag catcagtggc taccaagggc taggggcaggggaaggtgga 10680 gagttaatga ttaatagtat gaagtttcta tgtgagatga tgaaaatgttctggaaaaaa 10740 aaatatagtg gtgaggatgt agaatattgt gaatataatt aacggcatttaattgtacac 10800 ttaacatgat taatgtggca tattttatct tatgtatttg actacatccaagaaacactg 10860 ggagagggaa agcccaccat gtaaaataca cccaccctaa tcagatagtcctcattgtac 10920 ccaggtacag gcccctcatg acctgcacag gaataactaa ggatttaaggacatgaggct 10980 tcccagccaa ctgcaggtgc acaacataaa tgtatctgca aacagactgagagtaaagct 11040 gggggcacaa acctcagcac tgccaggaca cacacccttc tcgtggattctgactttatc 11100 tgacccggcc cactgtccag atcttgttgt gggattggga caagggaggtcataaagcct 11160 gtccccaggg cactctgtgt gagcacacga gacctcccca cccccccaccgttaggtctc 11220 cacacataga tctgaccatt aggcattgtg aggaggactc tagcgcgggctcagggatca 11280 caccagagaa tcaggtacag agaggaagac ggggctcgag gagctgatggatgacacaga 11340 gcagggttcc tgcagtccac aggtccagct caccctggtg taggtgccccatccccctga 11400 tccaggcatc cctgacacag ctccctcccg gagcctcctc ccaggtgacacatcagggtc 11460 cctcactcaa gctgtccaga gagggcagca ccttggacag cgcccaccccacttcactct 11520 tcctccctca cagggctcag ggctcagggc tcaagtctca gaacaaatggcagaggccag 11580 tgagcccaga gatggtgaca gggcaatgat ccaggggcag ctgcctgaaacgggagcagg 11640 tgaagccaca gatgggagaa gatggttcag gaagaaaaat ccaggaatgggcaggagagg 11700 agaggaggac acaggctctg tggggctgca gcccaggatg ggactaagtgtgaagacatc 11760 tcagcaggtg aggccaggtc ccatgaacag agaagcagct cccacctcccctgatgcacg 11820 gacacacaga gtgtgtggtg ctgtgccccc agagtcgggc tctcctgttctggtccccag 11880 ggagtgagaa gtgaggttga cttgtccctg ctcctctctg ctaccccaacattcaccttc 11940 tcctcatgcc cctctctctc aaatatgatt tggatctatg tccccgcccaaatctcatgt 12000 caaattgtaa accccaatgt tggaggtggg gccttgtgag aagtgattggataatgcggg 12060 tggattttct gctttgatgc tgtttctgtg atagagatct cacatgatctggttgtttaa 12120 aagtgtgtag cacctctccc ctctctctct ctctctctta ctcatgctctgccatgtaag 12180 acgttcctgt ttccccttca ccgtccagaa tgattgtaag ttttctgaggcctccccagg 12240 agcagaagcc actatgcttc ctgtacaact gcagaatgat gagcgaattaaacctctttt 12300 ctttataaat tacccagtct caggtatttc tttatagcaa tgcgaggacagactaataca 12360 atcttctact cccagatccc cgcacacgct tagccccaga catcactgcccctgggagca 12420 tgcacagcgc agcctcctgc cgacaaaagc aaagtcacaa aaggtgacaaaaatctgcat 12480 ttggggacat ctgattgtga aagagggagg acagtacact tgtagccacagagactgggg 12540 ctcaccgagc tgaaacctgg tagcactttg gcataacatg tgcatgacccgtgttcaatg 12600 tctagagatc agtgttgagt aaaacagcct ggtctggggc cgctgctgtccccacttccc 12660 tcctgtccac cagagggcgg cagagttcct cccaccctgg agcctccccaggggctgctg 12720 acctccctca gccgggccca cagcccagca gggtccaccc tcacccgggtcacctcggcc 12780 cacgtcctcc tcgccctccg agctcctcac acggactctg tcagctcctccctgcagcct 12840 atcggccgcc cacctgaggc ttgtcggccg cccacttgag gcctgtcggctgccctctgc 12900 aggcagctcc tgtcccctac accccctcct tccccgggct cagctgaaagggcgtctccc 12960 agggcagctc cctgtgatct ccaggacagc tcagtctctc acaggctccgacgcccccta 13020 tgctgtcacc tcacagccct gtcattacca ttaactcctc agtcccatgaagttcactga 13080 gcgcctgtct cccggttaca ggaaaactct gtgacaggga ccacgtctgtcctgctctct 13140 gtggaatccc agggcccagc ccagtgcctg acacggaaca gatgctccataaatactggt 13200 taaatgtgtg ggagatctct aaaaagaagc atatcacctc cgtgtggcccccagcagtca 13260 gagtctgttc catgtggaca caggggcact ggcaccagca tgggaggaggccagcaagtg 13320 cccgcggctg ccccaggaat gaggcctcaa cccccagagc ttcagaagggaggacagagg 13380 cctgcaggga atagatcctc cggcctgacc ctgcagccta atccagagttcagggtcagc 13440 tcacaccacg tcgaccctgg tcagcatccc tagggcagtt ccagacaaggccggaggtct 13500 cctcttgccc tccagggggt gacattgcac acagacatca ctcaggaaacggattcccct 13560 ggacaggaac ctggctttgc taaggaagtg gaggtggagc ctggtttccatcccttgctc 13620 caacagaccc ttctgatctc tcccacatac ctgctctgtt cctttctgggtcctatgagg 13680 accctgttct gccaggggtc cctgtgcaac tccagactcc ctcctggtaccaccatgggg 13740 aaggtggggt gatcacagga cagtcagcct cgcagagaca gagaccacccaggactgtca 13800 gggagaacat ggacaggccc tgagccgcag ctcagccaac agacacggagagggagggtc 13860 cccctggagc cttccccaag gacagcagag cccagagtca cccacctccctccaccacag 13920 tcctctcttt ccaggacaca caagacacct ccccctccac atgcaggatctggggactcc 13980 tgagacctct gggcctgggt ctccatccct gggtcagtgg cggggttggtggtactggag 14040 acagagggct ggtccctccc cagccaccac ccagtgagcc tttttctagcccccagagcc 14100 acctctgtca ccttcctgtt gggcatcatc ccaccttccc agagccctggagagcatggg 14160 gagacccggg accctgctgg gtttctctgt cacaaaggaa aataatccccctggtgtgac 14220 agacccaagg acagaacaca gcagaggtca gcactgggga agacaggttgtcctcccagg 14280 ggatgggggt ccatccacct tgccgaaaag atttgtctga ggaactgaaaatagaaggga 14340 aaaaagagga gggacaaaag aggcagaaat gagaggggag gggacagaggacacctgaat 14400 aaagaccaca cccatgaccc acgtgatgct gagaagtact cctgccctaggaagagactc 14460 agggcagagg gaggaaggac agcagaccag acagtcacag cagccttgacaaaacgttcc 14520 tggaactcaa gctcttctcc acagaggagg acagagcaga cagcagagaccatggagtct 14580 ccctcggccc ctccccacag atggtgcatc ccctggcaga ggctcctgctcacaggtgaa 14640 gggaggacaa cctgggagag ggtgggagga gggagctggg gtctcctgggtaggacaggg 14700 ctgtgagacg gacagagggc tcctgttgga gcctgaatag ggaagaggacatcagagagg 14760 gacaggagtc acaccagaaa aatcaaattg aactggaatt ggaaaggggcaggaaaacct 14820 caagagttct attttcctag ttaattgtca ctggccacta cgtttttaaaaatcataata 14880 actgcatcag atgacacttt aaataaaaac ataaccaggg catgaaacactgtcctcatc 14940 cgcctaccgc ggacattgga aaataagccc caggctgtgg agggccctgggaaccctcat 15000 gaactcatcc acaggaatct gcagcctgtc ccaggcactg gggtgcaaccaagatc 15056 15 858 DNA Artificial Sequence Mucin-TRE 15 cgagcggcccctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60 gggaggagctcctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120 cccaggtttactcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180 gagggaacccaggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240 cggggtgtggaacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300 ggggagggagcccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360 ccgctctgcttcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420 gcttcccgaccttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480 tagttgttgccctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540 ccccctcccccggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600 gggtagtcagggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660 gaggaggaagaggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720 ctgtgcctagggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780 gcccgctccacctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840 catttcaccaccaccatg 858 16 5224 DNA Artificial Sequence AlphaFP-TRE 16 gaattcttagaaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60 gagataagcattgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120 aacagactatgggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180 gttctatggggcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240 aagcccaaggtactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300 gagaaaatataaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360 tcatttgtatcaatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420 gagggtaggggtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480 tgcttgtgacaaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540 aagaagttaatggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600 acaagaggttccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660 ctcttctctagctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720 taagggttgggtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780 attttcaactaaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840 gtgagataagagaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900 gtatatatctaatatttaac actaacatca tgctaataat gataataatt actgtcattt 960 tttaatgtctataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020 acttgaggggatagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080 ttgcctgtcatacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140 ctttctaataccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200 tatgaaaaagtaaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260 acttaggactaaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320 acaaactctggttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380 ggtttctcttttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440 tgcaagcttatgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500 agtagtattcaaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560 caaaccggggagttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620 tcttgttccttaaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680 agagtgaaaagggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740 gtttgggtaaactggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800 agttactaagtctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860 ttgacagtattattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920 tgaaacaaaagagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980 ctataatagtcccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040 tgccaactttgccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100 ttcaaaactgcattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160 tttccaccaccttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220 tctaattttctttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280 cctccaaagatgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340 tgttgtgattactgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400 tacatggtgcctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460 gacttaacattttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520 agacccggttctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580 gaggtactatcactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640 aactcacccaaagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700 ttacgtgattaatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760 ctgctccgtaaggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820 cagaggaaaagtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880 gcatcgacatcttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940 ttatttgtagttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000 ttactaaaggcctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060 agatgatcagattggattac ttggccttga agatgatata tctacatcta tatctatatc 3120 tatatctatatctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180 aaatatgttttgtaaagtta taaagatttc agactttata gaatctggga tttgccaaat 3240 gtaacccctttctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300 aaatgttgctgagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360 catgcttgggcaagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420 aatacttcatgactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480 tatttgagtaaagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540 tggaatccaaacggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600 gcctgttttgttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660 aatggacaaaaactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720 tatactatgataaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780 taaatgtgtgccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840 tttaaaaattttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900 agatagaattaaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960 tccattatttctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020 gtacaatgatacacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080 acaatatattatgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140 ggctgtatatcaccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200 tttgattttataatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260 ggtgcctgggtctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320 ttgggaattcaaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380 tagacagggatgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440 catcttgagttgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500 taaaattattataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560 atacatcgcatactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620 tgtttctgagctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 4680 attattctgcaacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 4740 gttatatttgcaagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800 tgataggcatttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860 gctgtcttttctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920 tagggacaagtcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980 aaaataaaataagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040 ataaaatacaaataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100 ggaaataccataaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160 gaaaagagtataaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220 aaca 5224 17307 DNA Artificial Sequence Nucleotide sequence for ADP 17 gatgaccggctcaaccatcg cgcccacaac ggactatcgc aacaccactg ctaccggact 60 aacatctgccctaaatttac cccaagttca tgcctttgtc aatgactggg cgagcttgga 120 catgtggtggttttccatag cgcttatgtt tgtttgcctt attattatgt ggcttatttg 180 ttgcctaaagcgcagacgcg ccagaccccc catctatagg cctatcattg tgctcaaccc 240 acacaatgaaaaaattcata gattggacgg tctgaaacca tgttctcttc ttttacagta 300 tgattaa 30718 101 PRT Artificial Sequence Amino acid sequence for ADP 18 Met ThrGly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr 1 5 10 15 AlaThr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln Val His Ala Phe 20 25 30 ValAsn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu 35 40 45 MetPhe Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg 50 55 60 ArgArg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro 65 70 75 80His Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu 85 90 95Leu Leu Gln Tyr Asp 100 19 28 DNA Artificial Sequence PCR EMCV IRES (PCRprimer 96.74.2) 19 gacgtcgact aattccggtt attttcca 28 20 28 DNAArtificial Sequence PCR EMCV IRES (PCR primer 96.74.1) 20 gacgtcgacatcgtgttttt caaaggaa 28 21 25 DNA Artificial Sequence Ad5 sequence to1314 to 1338 (PCR primer 96.74.3) 21 cctgagacgc ccgacatcac ctgtg 25 2230 DNA Artificial Sequence Antisense of Ad5 sequence 1572 to 1586 (PCRprimer 96.74.6) 22 gtcgaccatt cagcaaacaa aggcgttaac 30 23 30 DNAArtificial Sequence Ad5 sequence 1714 to 1728 (PCR primer 96.74.4) 23tgctgaatgg tcgacatgga ggcttgggag 30 24 26 DNA Artificial SequenceAntisense of Ad5 sequence 2070 to 2094 (PCR primer 96.74.5) 24cacaaaccgc tctccacaga tgcatg 26 25 29 DNA Artificial Sequence Human UPII(PCR primer 127.2.1) 25 aggaccggtc actatagggc acgcgtggt 29 26 31 DNAArtificial Sequence Human UPII (PCR primer 127.2.2) 26 aggaccggtgggatgctggg ctgggaggtg g 31 27 29 DNA Artificial Sequence PCR primer100.113.1 27 aggggtaccc actatagggc acgcgtggt 29 28 32 DNA ArtificialSequence PCR primer 100.113.2 28 acccaagctt gggatgctgg gctgggaggt gg 3229 30 DNA Artificial Sequence PCR primer 127.50.1 29 aggaccggtcaggcttcacc ccagacccac 30 30 24 DNA Artificial Sequence PCR primer31.166.1 30 tgcgccggtg tacacaggaa gtga 24 31 21 DNA Artificial SequencePCR primer 32.32.1 31 gagtttgtgc catcggtcta c 21 32 21 DNA ArtificialSequence PCR primer 32.32.2 32 aatcaatcct tagtcctcct g 21 33 25 DNAArtificial Sequence PCR primer 51.176 33 gcagaaaaat cttccaaaca ctccc 2534 27 DNA Artificial Sequence PCR primer 99.120.1 34 acgtacaccggtcgttacat aacttac 27 35 26 DNA Artificial Sequence PCR primer 99.120.235 ctagcaaccg gtcggttcac taaacg 26

What is claimed is:
 1. A replication-competent adenovirus vectorcomprising first and second genes co-transcribed as a single mRNAwherein the first and the second genes are under transcriptional controlof a heterologous, target cell-specific transcriptional regulatoryelement (TRE), wherein the second gene has a mutation in or deletion ofits endogenous promoter and is under translational control of aninternal ribosome entry site (IRES) and wherein said vector exhibitsgreater specificity for the target cell than an adenovirus vectorcomprising a target cell-specific TRE operably linked to a gene andlacking an IRES.
 2. The vector of claim 1, wherein at least one of saidfirst and second genes is an adenovirus gene.
 3. The vector of claim 2,wherein both of said first and said second genes are adenovirus genes.4. The vector of claim 2, wherein at least one of said first and saidsecond adenovirus gene is essential for viral replication.
 5. The vectorof claim 4, wherein the adenovirus gene essential for viral-replicationis an adenovirus early gene.
 6. The vector of claim 5, wherein theadenovirus early gene includes E1A, E1B, E2, or E4.
 7. The vector ofclaim 4, wherein the adenovirus gene essential for viral replication isan adenovirus late gene.
 8. The vector of claim 3, wherein both saidfirst and said second adenovirus genes are essential for viralreplication.
 9. The vector of claim 8, wherein at least one of saidfirst and said second adenovirus genes is an adenovirus early gene. 10.The vector of claim 8, wherein at least one of said first and saidsecond adenovirus genes is an adenovirus late gene.
 11. The vector ofclaim 8, wherein said first adenovirus gene is E1A and said secondadenovirus gene is E1B.
 12. The vector of claim 11 wherein E1A has itsendogenous promoter deleted.
 13. The vector of claim 11 wherein E1A hasan inactivation of E1A enhancer I.
 14. The vector of claim 11 whereinE1B has an inactivation of its endogenous promoter.
 15. The vector ofclaim 11 wherein E1B has a deletion of the 19-kDa region.
 16. The vectorof claim 11 wherein E1A has an inactivation of its endogenous promoterand E1B has an inactivation of its endogenous promoter.
 17. The vectorof claim 16 wherein E1B has a deletion of the 19-kDa region.
 18. Thevector of claim 16 wherein E1A has an inactivation of E1A enhancer I.19. The vector of claim 1, wherein the internal ribosome entry site(IRES) is from EMCV.
 20. The vector of claim 1 wherein the IRES is fromVEGF.
 21. The vector of claim 1 wherein the IRES includes the 5′UTR ofHCV; the 5′ UTR of BiP; or the 5′UTR of PDGF.
 22. The vector of claim 1,wherein the TRE is specific for a target cell that is a cancer cell. 23.The vector of claim 22 wherein the cancer cell includes a prostatecancer cell, a breast cancer cell, a hepatoma cell, a melanoma cell, abladder cell or a colon cancer cell.
 24. The vector of claim 22, whereinthe TRE includes the probasin (PB) TRE, the prostate-specific antigen(PSA) TRE, the mucin (MUC1) TRE, the α-fetoprotein (AFP) TRE, the hKLK2TRE, the tyrosinase TRE, the human uroplakin II (hUPII) TRE or thecarcinoembryonic antigen (CEA) TRE.
 25. The vector of claim 9 whereinsaid first adenovirus gene has a deletion of its endogenous promoter.26. The vector of claim 25 wherein said first adenovirus gene is E1A.27. The vector of claim 9 wherein said first and/or said secondadenovirus gene has a deletion of an enhancer region.
 28. The vector ofclaim 27 wherein said first gene is E1A and said enhancer is E1Aenhancer I.
 29. The vector of claim 1 wherein said TRE has an endogenoussilencer element deleted.
 30. The vector of claim 1 wherein saidadenovirus vector comprises an E3 region.
 31. The adenovirus vector ofclaim 11 wherein said adenovirus comprises an E3 region.
 32. Theadenovirus vector of claim 11 further comprising a transgene.
 33. Anadenovirus vector comprising a gene under transcriptional control of amelanocyte-specific TRE.
 34. The vector of claim 33 wherein said gene isan adenoviral gene.
 35. The vector of claim 34 wherein said adenoviralgene is a gene essential for replication.
 36. The vector of claim 32wherein said transgene is co-transcribed with said first and said secondgene and said transgene is under the translation control of a separateinternal ribosome entry site (IRES).
 37. The vector of claim 36 whereinsaid IRES is from EMCV.
 38. The vector of claim 36 wherein said IRES isfrom VEGF.
 39. The vector of claim 30 further comprising an adenovirusdeath protein gene (ADP).
 40. The vector of claim 32 wherein saidtransgene is a cytotoxic gene.
 41. The vector of claim 1 wherein saidfirst adenovirus gene is essential for viral replication and said secondadenovirus gene is the adenovirus death protein gene (ADP).
 42. Thevector of claim 41 wherein said first adenovirus gene is E1A.
 43. Thevector of claim 42 wherein E1A has a deletion of its endogenouspromoter.
 44. The vector of claim 42 wherein said E1A has a deletion ofE1A enhancer I.
 45. The vector of claim 1 wherein said first gene isessential for viral replication and said second gene is E3.
 46. Thevector of claim 45 wherein said first gene is E1A.
 47. A compositioncomprising a vector according to claim
 1. 48. The composition of claim47 further comprising a pharmaceutically acceptable excipient.
 49. Acomposition comprising a vector according to claim
 30. 50. Thecomposition of claim 49 further comprising a pharmaceutically acceptableexcipient.
 51. A host cell comprising the vector of claim
 1. 52. A hostcell comprising the vector of claim
 30. 53. An adenovirus vectorcomprising E1B under transcriptional control of a heterologous, targetcell specific TRE, wherein E1B has a deletion of part or all of the19-kDa region.
 54. A host cell comprising the adenovirus vector of claim53.
 55. A method for propagating a replication-competent adenovirusvector comprising a target cell-specific TRE, said method comprisingcombining an adenovirus vector of claim 1 with mammalian cells thatpermit the function of a target cell-specific TRE, such that theadenovirus vector enters the cell, whereby said adenovirus vector ispropagated.
 56. A method for conferring selective cytotoxicity in targetcells, comprising contacting the cells with an adenovirus vector ofclaim 41 whereby the vector enters the cell.
 57. A method for modifyingthe genotype of a target cell, comprising contacting the cell with anadenovirus vector of claim 1, wherein the vector enters the cell.
 58. Amethod for suppressing tumor cell growth, comprising contacting a tumorcell with an adenovirus vector of claim 41 such that the adenovirusvector enters the tumor cell and exhibits selective cytotoxicity for thetumor cell.